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Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported. In the primary analysis of the global phase II ELIANA trial (ClinicalTrials.gov identifier: NCT02435849 ), tisagenlecleucel provided an overall remission rate of 81% in pediatric and young adult patients with relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), with 59% of responders remaining relapse-free at 12 months. Here, we report an update on efficacy, safety, and patient-reported quality of life in 79 pediatric and young adult patients with R/R B-ALL following a median follow-up of 38.8 months. The overall remission rate was 82%. The median event-free survival was 24 months, and the median overall survival was not reached. Event-free survival was 44% (95% CI, 31 to 57) and overall survival was 63% (95% CI, 51 to 73) at 3 years overall (most events occur within the first 2 years). The estimated 3-year relapse-free survival with and without censoring for subsequent therapy was 52% (95% CI, 37 to 66) and 48% (95% CI, 34 to 60), respectively. No new or unexpected long-term adverse events were reported. Grade 3/4 adverse events were reported in 29% of patients > 1 year after infusion; grade 3/4 infection rate did not increase > 1 year after infusion. Patients reported improvements in quality of life up to 36 months after infusion. These findings demonstrate favorable long-term safety and suggest tisagenlecleucel as a curative treatment option for heavily pretreated pediatric and young adult patients with R/R B-ALL.

Purpose: To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell–associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment. Experimental Design: We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials. Results: We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS. Conclusions: We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS.

Down syndrome-associated acute lymphoblastic leukemia (DS-ALL) patients suffer risk of chemotherapy-associated toxicities and poor outcomes. We evaluated tisagenlecleucel in 16 patients with DS-ALL in two phase 2 trials (ELIANA [NCT02435849], ENSIGN [NCT02228096]) and a phase 3b, managed access protocol (B2001X [NCT03123939]). Patients were 5-22 years old, had a median of two prior lines of therapy (range, 1-4), and four (25%) had prior stem cell transplants. Fourteen of 16 patients (88%) achieved complete remission (CR) or CR with incomplete blood count recovery (CRi); 12 of 14 (86%) with CR/CRi were minimal residual disease-negative. With a median follow-up of 13.2 months (range, 0.5-49.3 months), six patients (43%) relapsed after CR (three, CD19-negative; three, unknown) between 80-721 days post-infusion. Ongoing remissions in nine patients ranged from 6-48 months. Any-grade and grade 3/4 AEs occurred in 16 and 14 patients, respectively; 44% experienced grade 3/4 cytokine release syndrome and 13% experienced grade 3/4 neurological events. Grade 3/4 prolonged cytopenias occurred in 44% of patients. No grade 3/4 infections were observed. Tisagenlecleucel expansion and long-term persistence were consistent with previous reports. Comparable to ALL patients without DS, tisagenlecleucel produced high remission rates, manageable side-effects, and promising long-term outcomes in pediatric/young adult patients with DS-ALL.Children with Down syndrome have a 20 times higher risk of developing a type of blood cancer called Down syndrome-associated acute lymphoblastic leukemia (ALL). Children who develop Down syndrome-associated ALL typically receive chemotherapy to treat their cancer; however, they can experience severe toxicity or other consequences from these therapies, especially stem cell transplant, and have a poor prognosis if their disease returns after treatment. These children need an effective but less toxic treatment option. Tisagenlecleucel is a chimeric antigen receptor-T cell therapy that specially modifies the patient’s own T-cells to recognize and attack the cancer cells. Tisagenlecleucel is approved for use in children and young adults with ALL whose disease reappears after two or more treatments or whose disease doesn’t respond to treatment. Here we present data from 16 patients across three clinical studies showing that tisagenlecleucel is well-tolerated and an effective treatment option for children and young adults with Down syndrome-associated ALL, and was similar to what is observed in patients without Down syndrome. Taken together, patients with Down syndrome-associated ALL have unique medical needs, and tisagenlecleucel may help them live longer, avoid stem cell transplantation, and the toxicity from chemotherapy.

BackgroundImmunocompromised patients are at increased risk of SARS-CoV-2 infections. Patients undergoing chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory B-cell malignancies are uniquely immunosuppressed due to CAR T-mediated B-cell aplasia (BCA). While SARS-CoV-2 mortality rates of 33%–40% are reported in adult CAR T-cell recipients, outcomes in pediatric and young adult CAR T-cell recipients are limited.MethodsWe created an international retrospective registry of CAR T recipients aged 0–30 years infected with SARS-CoV-2 within 2 months prior to or any time after CAR T infusion. SARS-CoV-2-associated illness was graded as asymptomatic, mild, moderate, or severe COVID-19, or multisystem inflammatory syndrome in children (MIS-C). To assess for risk factors associated with significant SARS-CoV-2 infections (infections requiring hospital admission for respiratory distress or supplemental oxygen), univariate and multivariable regression analyses were performed.ResultsNine centers contributed 78 infections in 75 patients. Of 70 SARS-CoV-2 infections occurring after CAR T infusion, 13 (18.6%) were classified as asymptomatic, 37 (52.9%) mild, 11 (15.7%) moderate, and 6 (8.6%) severe COVID-19. Three (4.3%) were classified as MIS-C. BCA was not significantly associated with infection severity. Prior to the emergence of the Omicron variant, of 47 infections, 19 (40.4%) resulted in hospital admission and 7 (14.9%) required intensive care, while after the emergence of the Omicron variant, of 23 infections, only 1 (4.3%) required admission and the remaining 22 (95.7%) had asymptomatic or mild COVID-19. Death occurred in 3 of 70 (4.3%); each death involved coinfection or life-threatening condition. In a multivariable model, factors associated with significant SARS-CoV-2 infection included having two or more comorbidities (OR 7.73, CI 1.05 to 74.8, p=0.048) and age ≥18 years (OR 9.51, CI 1.90 to 82.2, p=0.014). In the eight patients infected with SARS-CoV-2 before CAR T, half of these patients had their CAR T infusion delayed by 15–30 days.ConclusionsIn a large international cohort of pediatric and young adult CAR-T recipients, SARS-CoV-2 infections resulted in frequent hospital and intensive care unit admissions and were associated with mortality in 4.3%. Patients with two or more comorbidities or aged ≥18 years were more likely to experience significant illness. Suspected Omicron infections were associated with milder disease.

The seventh multi-stakeholder Paediatric Strategy Forum focused on chimeric antigen receptor (CAR) T-cells for children and adolescents with cancer. The development of CAR T-cells for patients with haematological malignancies, especially B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), has been spectacular. However, currently, there are scientific, clinical and logistical challenges for use of CAR T-cells in BCP-ALL and other paediatric malignancies, particularly in acute myeloid leukaemia (AML), lymphomas and solid tumours. The aims of the Forum were to summarise the current landscape of CAR T-cell therapy development in paediatrics, too identify current challenges and future directions, with consideration of other immune effector modalities and ascertain the best strategies to accelerate their development and availability to children. Although the effect is of limited duration in about half of the patients, anti-CD19 CAR T-cells produce high response rates in relapsed/refractory BCP-ALL and this has highlighted previously unknown mechanisms of relapse. CAR T-cell treatment as first- or second-line therapy could also potentially benefit patients whose disease has high-risk features associated with relapse and failure of conventional therapies. Identifying patients with very early and early relapse in whom CAR T-cell therapy may replace haematopoietic stem cell transplantation and be definitive therapy versus those in whom it provides a more effective bridge to haematopoietic stem cell transplantation is a very high priority. Development of approaches to improve persistence, either by improving T cell fitness or using more humanised/fully humanised products and co-targeting of multiple antigens to prevent antigen escape, could potentially further optimise therapy. Many differences exist between paediatric B-cell non-Hodgkin lymphomas (B-NHL) and BCP-ALL. In view of the very small patient numbers with relapsed lymphoma, careful prioritisation is needed to evaluate CAR T-cells in children with Burkitt lymphoma, primary mediastinal B cell lymphoma and other NHL subtypes. Combination trials of alternative targets to CD19 (CD20 or CD22) should also be explored as a priority to improve efficacy in this population. Development of CD30 CAR T-cell immunotherapy strategies in patients with relapsed/refractory Hodgkin lymphoma will likely be most efficiently accomplished by joint paediatric and adult trials. CAR T-cell approaches are early in development for AML and T-ALL, given the unique challenges of successful immunotherapy actualisation in these diseases. At this time, CD33 and CD123 appear to be the most universal targets in AML and CD7 in T-ALL. The results of ongoing or planned first-in-human studies are required to facilitate further understanding. There are promising early results in solid tumours, particularly with GD2 targeting cell therapies in neuroblastoma and central nervous system gliomas that represent significant unmet clinical needs. Further understanding of biology is critical to success. The comparative benefits of autologous versus allogeneic CAR T-cells, T-cells engineered with T cell receptors T-cells engineered with T cell receptor fusion constructs, CAR Natural Killer (NK)-cell products, bispecific T-cell engager antibodies and antibody-drug conjugates require evaluation in paediatric malignancies. Early and proactive academia and multi-company engagement are mandatory to advance cellular immunotherapies in paediatric oncology. Regulatory advice should be sought very early in the design and preparation of clinical trials of innovative medicines, for which regulatory approval may ultimately be sought. Aligning strategic, scientific, regulatory, health technology and funding requirements from the inception of a clinical trial is especially important as these are very expensive therapies. The model for drug development for cell therapy in paediatric oncology could also involve a ‘later stage handoff’ to industry after early development in academic hands. Finally, and very importantly, strategies must evolve to ensure appropriate ease of access for children who need and could potentially benefit from these therapies.

Despite improved outcomes achieved in the last decades for children with newly diagnosed leukaemia and lymphoma, treatment of patients with refractory/relapsed disease remains a challenge. The cure rate is still unsatisfactory and often achieved at the cost of significant morbidity. Exploring treatment with novel agents should offer less toxic therapeutic options, without compromising efficacy. Bispecific and antibody–drug conjugates targeting CD19 and CD22 (blinatumomab and inotuzumab ozogamicin) play an important role in the treatment of relapsed and refractory B-cell precursor acute lymphoblastic leukaemia (BCP-ALL); antibodies targeting CD123 and CD38 are also under investigation for acute myeloid leukaemia (AML) and T-ALL, respectively. Targeted therapy with small molecules is of primary importance for specific genetic subtypes, such as BCR-ABL-positive ALL, FLT3-ITD AML and anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma. KMT2A-directed targeted therapy with menin inhibitors holds promise to be of relevance in KMT2A-rearranged leukaemias, known to have dismal prognosis. Target inhibition in cellular pathways such as BCL-2, RAS, MEK, Bruton's tyrosine kinase, JAK-STAT or CDK4/CDK6 inhibition may be suitable for different diseases with common mutated pathways. Nevertheless, development and approval of new agents for paediatric cancers lags behind adult therapeutic options. New regulations were implemented to accelerate drug development for children. Considering the number of oncology medicinal products available for adults and the rarity of paediatric cancers, prioritisation based on scientific evidence and medical need, as well as international collaboration, is critical. Herein, we review the current status of drug development for children with leukaemia and lymphoma, excluding cellular therapy despite its well-known significance.

Chimeric antigen receptor T-cell (CAR-T) therapy is an exciting development in the field of cancer immunology and has received a lot of interest in recent years. Many time-to-event (TTE) endpoints related to relapse, disease progression, and remission are analyzed in CAR-T studies to assess treatment efficacy. Definitions of these TTE endpoints are not always consistent, even for the same outcomes (e.g., progression-free survival), which often stems from analysis choices regarding which events to consider as part of the composite endpoint, censoring or competing risk in the analysis. Subsequent therapies such as hematopoietic stem cell transplantation are common but are not treated the same in different studies. Standard survival analysis methods are commonly applied to TTE analyses but often without full consideration of the assumptions inherent in the chosen analysis. We highlight two important issues of TTE analysis that arise in CAR-T studies, as well as in other settings in oncology: the handling of competing risks and assessing the association between a time-varying (post-infusion) exposure and the TTE outcome. We review existing analytical methods, including the cumulative incidence function and regression models for analysis of competing risks, and landmark and time-varying covariate analysis for analysis of post-infusion exposures. We clarify the scientific questions that the different analytical approaches address and illustrate how the application of an inappropriate method could lead to different results using data from multiple published CAR-T studies. Codes for implementing these methods in standard statistical software are provided.

Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. Significance: Resistance to CART19 is a significant barrier to efficacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigen-independent resistance to CAR therapy. See related commentary by Green and Neelapu, p. 492.

PURPOSE The anti-CD19 chimeric antigen receptor T-cell therapy tisagenlecleucel (CTL019) has an 81% response rate in children with relapsed or chemotherapy refractory (r/r) B-cell acute lymphoblastic leukemia (ALL). Cytokine release syndrome (CRS) is a life-threatening treatment-related toxicity that limits the full therapeutic potential in adults. We report outcomes for adults with r/r ALL treated with an optimized CTL019 dosing and CRS management strategy. METHODS Adults with r/r B-cell ALL received CTL019 in 1 of 2 trials. Patients received lymphodepletion followed by CTL019 as either a one-time infusion or fractionated infusions split over 3 days (day 1, 10%; day 2, 30%; day 3, 60%), which allowed for day 2 and day 3 doses to be held for early CRS. Total planned CTL019 dose varied with adaptive protocol modifications in response to efficacy and CRS toxicity. RESULTS Thirty-five adults with r/r ALL received CTL019 in 1 of 3 dosing cohorts. The low-dose cohort (n = 9) received single or fractionated dosing and had manageable toxicity with a 33% complete remission (CR) rate. In the high-dose single infusion cohort, 3 of 6 patients with refractory CRS concurrent with culture-positive sepsis died, and 3 achieved CR. The 20 patients in the high-dose fractionated (HDF) cohort had a 90% CR rate and manageable CRS. The HDF cohort had the highest survival, with a 2-year overall survival of 73% (95% CI, 46% to 88%) and event-free survival of 49.5% (95% CI, 21% to 73%). CONCLUSION Fractionated dosing of CTL019 with intrapatient dose modification optimizes safety without compromising efficacy in adults with r/r ALL.

Chimeric antigen receptor–engineered T cells targeting CD19 (CART19) provide an effective treatment for pediatric acute lymphoblastic leukemia but are less effective for chronic lymphocytic leukemia (CLL), focusing attention on improving efficacy. CART19 harbor an engineered receptor, which is delivered through lentiviral vector integration, thereby marking cell lineages and modifying the cellular genome by insertional mutagenesis. We recently reported that vector integration within the host TET2 gene was associated with CLL remission. Here, we investigated clonal population structure and therapeutic outcomes in another 39 patients by high-throughput sequencing of vector-integration sites. Genes at integration sites enriched in responders were commonly found in cell-signaling and chromatin modification pathways, suggesting that insertional mutagenesis in these genes promoted therapeutic T cell proliferation. We also developed a multivariate model based on integration-site distributions and found that data from preinfusion products forecasted response in CLL successfully in discovery and validation cohorts and, in day 28 samples, reported responders to CLL therapy with high accuracy. These data clarify how insertional mutagenesis can modulate cell proliferation in CART19 therapy and how data on integration-site distributions can be linked to treatment outcomes.

PURPOSE CD19-targeted chimeric antigen receptor (CAR)–modified T cells demonstrate unprecedented responses in B-cell acute lymphoblastic leukemia (B-ALL); however, relapse remains a substantial challenge. Short CAR T-cell persistence contributes to this risk; therefore, strategies to improve persistence are needed. METHODS We conducted a pilot clinical trial of a humanized CD19 CAR T-cell product (huCART19) in children and young adults with relapsed or refractory B-ALL (n = 72) or B-lymphoblastic lymphoma (n = 2), treated in two cohorts: with (retreatment, n = 33) or without (CAR-naive, n = 41) prior CAR exposure. Patients were monitored for toxicity, response, and persistence of huCART19. RESULTS Seventy-four patients 1-29 years of age received huCART19. Cytokine release syndrome developed in 62 (84%) patients and was grade 4 in five (6.8%). Neurologic toxicities were reported in 29 (39%), three (4%) grade 3 or 4, and fully resolved in all cases. The overall response rate at 1 month after infusion was 98% (100% in B-ALL) in the CAR-naive cohort and 64% in the retreatment cohort. At 6 months, the probability of losing huCART19 persistence was 27% (95% CI, 14 to 41) for CAR-naive and 48% (95% CI, 30 to 64) for retreatment patients, whereas the incidence of B-cell recovery was 15% (95% CI, 6 to 28) and 58% (95% CI, 33 to 77), respectively. Relapse-free survival at 12 and 24 months, respectively, was 84% (95% CI, 72 to 97) and 74% (95% CI, 60 to 90) in CAR-naive and 74% (95% CI, 56 to 97) and 58% (95% CI, 37 to 90) in retreatment cohorts. CONCLUSION HuCART19 achieved durable remissions with long-term persistence in children and young adults with relapsed or refractory B-ALL, including after failure of prior CAR T-cell therapy.

7013 Background: CD19-directed chimeric antigen receptor T cell (CART) therapy has dramatically improved survival for children with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). While significant socioeconomic (SES) outcome disparities exist for children with newly diagnosed B-ALL, the impact of SES on CART access and outcomes is poorly described. Using the largest single-center pediatric CART experience, we investigated the hypothesis that poverty-exposed children would have inferior survival outcomes compared to unexposed children. Methods: Retrospective cohort study of US pediatric patients treated on CD19 CART clinical trials or with commercial tisagenlecleucel at Children's Hospital of Philadelphia from 2012-2020. Poverty was the primary exposure, defined at the household-level by insurance status (public vs private). Neighborhood opportunity was defined by census-derived Childhood Opportunity Index (COI) (low [q1-2] vs high [q3-4]). Overall survival (OS) and relapse-free survival (RFS) were evaluated by Kaplan Meier methods, and association with exposures by Cox regression models. Results: Among 206 patients, 36% were household poverty exposed, 24.9% low COI, 21.4% identified as Hispanic, 7.3% non-Hispanic Black, 63.6% non-Hispanic White, and 7.7% non-Hispanic Other. Household-poverty exposure was similar between local and referred patients (32.4% vs 36.7%). Patients unexposed to poverty at the household level or with high COI presented to CART with high disease burden (37.1% vs 26%, p = 0.049, 37.9% vs. 29.7%, p = 0.002). In multivariate analysis adjusting for age, race/ethnicity, disease burden, relapse status, and inotuzumab exposure, there were no significant differences in OS by householdverty (HR 0.86, 95%CI 0.50-1.48, p = 0.575) or low COI (HR 1.03, 95%CI 0.53-1.99, p = 0.932). Low COI was associated with inferior RFS (HR 2.26, 95%CI 1.34-8.80, p = 0.002). There was no significant difference in RFS by household-poverty (HR 0.84, 95%CI 0.48-1.44, p = 0.520). Conclusions: Household poverty was not associated with inferior survival outcomes in pediatric patients who received CART for r/r B-ALL. Patients with low neighborhood opportunity had increased hazard of relapse, a finding that requires investigation of the COI components underlying this association. Patients of higher proxied SES were more likely to have high disease burden, an access inequity potentially reflecting referral pattern bias or greater ability of advantaged families to advocate for CART. Future institutional and multi-center studies should utilize patient-reported social determinants of health to investigate mechanisms driving these disparities and guide care delivery interventions to improve equity in access and outcomes. Clinical trials: NCT01626495, NCT02435849, NCT02374333, NCT02228096, NCT02906371

Purpose: Tisagenlecleucel is an anti-CD19 chimeric antigen receptor (CAR19) T-cell therapy approved for the treatment of children and young adults with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL). Patients and Methods: We evaluated the cellular kinetics of tisagenlecleucel, the effect of patient factors, humoral immunogenicity, and manufacturing attributes on its kinetics, and exposure-response analysis for efficacy, safety and pharmacodynamic endpoints in 79 patients across two studies in pediatric B-ALL (ELIANA and ENSIGN). Results: Using quantitative polymerase chain reaction to quantify levels of tisagenlecleucel transgene, responders (N = 62) had ≈2-fold higher tisagenlecleucel expansion in peripheral blood than nonresponders (N = 8; 74% and 104% higher geometric mean Cmax and AUC0-28d, respectively) with persistence measurable beyond 2 years in responding patients. Cmax increased with occurrence and severity of cytokine release syndrome (CRS). Tisagenlecleucel continued to expand and persist following tocilizumab, used to manage CRS. Patients with B-cell recovery within 6 months had earlier loss of the transgene compared with patients with sustained clinical response. Clinical responses were seen across the entire dose range evaluated (patients ≤50 kg: 0.2 to 5.0 × 106/kg; patients >50 kg: 0.1 to 2.5 × 108 CAR-positive viable T cells) with no relationship between dose and safety. Neither preexisting nor treatment-induced antimurine CAR19 antibodies affected the persistence or clinical response. Conclusions: Response to tisagenlecleucel was associated with increased expansion across a wide dose range. These results highlight the importance of cellular kinetics in understanding determinants of response to chimeric antigen receptor T-cell therapy.

PURPOSETo prospectively evaluate the effectiveness of risk-adapted preemptive tocilizumab (PT) administration in preventing severe cytokine release syndrome (CRS) after CTL019, a CD19 chimeric antigen receptor T-cell therapy.METHODSChildren and young adults with CD19-positive relapsed or refractory B-cell acute lymphoblastic leukemia were assigned to high- (≥ 40%) or low- (< 40%) tumor burden cohorts (HTBC or LTBC) based on a bone marrow aspirate or biopsy before infusion. HTBC patients received a single dose of tocilizumab (8-12 mg/kg) after development of high, persistent fevers. LTBC patients received standard CRS management. The primary end point was the frequency of grade 4 CRS (Penn scale), with an observed rate of ≤ 5 of 15 patients in the HTBC pre-defined as clinically meaningful. In post hoc analyses, the HTBC was compared with a historical cohort of high-tumor burden patients from the initial phase I CTL019 trial.RESULTSThe primary end point was met. Seventy patients were infused with CTL019, 15 in the HTBC and 55 in the LTBC. All HTBC patients received the PT intervention. The incidence of grade 4 CRS was 27% (95% CI, 8 to 55) in the HTBC and 3.6% (95% CI, 0.4 to 13) in the LTBC. The best overall response rate was 87% in the HTBC and 100% in the LTBC. Initial CTL019 expansion was greater in the HTBC than the LTBC ( P < .001), but persistence was not different ( P = .73). Event-free and overall survival were worse in the HTBC ( P = .004, P < .001, respectively). In the post hoc analysis, grade 4 CRS was observed in 27% versus 50% of patients in the PT and prior phase I cohorts, respectively ( P = .18).CONCLUSIONRisk-adapted PT administration resulted in a decrease in the expected incidence of grade 4 CRS, meeting the study end point, without adversely impacting the antitumor efficacy or safety of CTL019.

Immune effector cell (IEC) therapies offer durable and sustained remissions in significant numbers of patients with hematological cancers. While these unique immunotherapies have improved outcomes for pediatric and adult patients in a number of disease states, as ‘living drugs,’ their toxicity profiles, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), differ markedly from conventional cancer therapeutics. At the time of article preparation, the US Food and Drug Administration (FDA) has approved tisagenlecleucel, axicabtagene ciloleucel, and brexucabtagene autoleucel, all of which are IEC therapies based on genetically modified T cells engineered to express chimeric antigen receptors (CARs), and additional products are expected to reach marketing authorization soon and to enter clinical development in due course. As IEC therapies, especially CAR T cell therapies, enter more widespread clinical use, there is a need for clear, cohesive recommendations on toxicity management, motivating the Society for Immunotherapy of Cancer (SITC) to convene an expert panel to develop a clinical practice guideline. The panel discussed the recognition and management of common toxicities in the context of IEC treatment, including baseline laboratory parameters for monitoring, timing to onset, and pharmacological interventions, ultimately forming evidence- and consensus-based recommendations to assist medical professionals in decision-making and to improve outcomes for patients.

Background T-ALL/LBL represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 85% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage regimens induce remissions in only ~20-30% of cases, and survival is dismal. T-ALL/LBL is a genetically diverse group, but with universal overexpression of CD7, making this a suitable target for immunotherapy. Despite the success of CAR-T cells in B-cell malignancies, CAR-T cell development in T-cell malignancies has proven challenging due to fratricide and high risk of contamination of the genetically modified CAR-T product with the patient's malignant T cells. WU-CART-007 is a CD7-directed, genetically modified, allogeneic, 'off the shelf', fratricide-resistant chimeric antigen receptor (CAR) T-cell product for the treatment of CD7+ve hematologic malignancies. Methods This multicenter, open-label, dose-escalation, Phase 1/2 study (NCT#04984356) of WU-CART-007 in patients ≥ 12 years old, with relapsed or refractory T-ALL/LBL is designed to characterize the safety, tolerability, dose-limiting toxicities (DLTs), and maximum tolerated dose (MTD)/maximum administered dose (MAD; if no MTD defined) (Phase 1), and to investigate the preliminary anti-tumor activity, as measured by objective response rate (ORR) and duration of response (DOR) (Phase 2). Phase 1 is comprised of a dose escalation segment and will proceed according to a standard 3+3 design testing up to 4 dose levels from 1 to 9 x 10 8 cells. Adolescent patients, ages 12-17, will be eligible for enrollment in Phase 1 Dose Escalation beginning at Dose level 3 and 4, and during Phase 2 Cohort Expansion. Upon reaching the MTD and/or RP2D, the Phase 2 portion comprised of the cohort expansion segment will be launched. A Simon's optimal two-stage design will be implemented to enroll patients (an interim analysis for futility in the first stage and the final analysis in the second stage) for Phase 2 dose expansion cohort to confirm safety and explore preliminary efficacy. All patients will receive a single infusion of WU-CART-007 cells on day 1 following a lymphodepleting conditioning therapy consisting of fludarabine and cyclophosphamide on days -5 to -3. Patients will be hospitalized for a minimum of 7 days following WU-CART-007 administration. Response will be assessed on Cycle 1 Day 28 (± 1 days), and at Months 3, 6, 12, and 24, by bone marrow aspirate and biopsy and PET/CT if indicated. Response will be defined as per modified NCCN Guidelines Version 2.2020. Disclosures Ghobadi: Atara: Consultancy; Amgen: Consultancy, Research Funding; Wugen: Consultancy; Celgene: Consultancy; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding. Locke: Janssen: Consultancy, Other: Scientific Advisory Role; Kite, a Gilead Company: Consultancy, Other: Scientific Advisory Role, Research Funding; Iovance Biotherapeutics: Consultancy, Other: Scientific Advisory Role; Legend Biotech: Consultancy, Other; Novartis: Consultancy, Other, Research Funding; Takeda: Consultancy, Other; Wugen: Consultancy, Other; Cowen: Consultancy; Umoja: Consultancy, Other; Bluebird Bio: Consultancy, Other: Scientific Advisory Role; Calibr: Consultancy, Other: Scientific Advisory Role; BMS/Celgene: Consultancy, Other: Scientific Advisory Role; GammaDelta Therapeutics: Consultancy, Other: Scientific Advisory Role; Cellular Biomedicine Group: Consultancy, Other: Scientific Advisory Role; Amgen: Consultancy, Other: Scientific Advisory Role; Allogene Therapeutics: Consultancy, Other: Scientific Advisory Role, Research Funding; EcoR1: Consultancy; Emerging Therapy Solutions: Consultancy; Gerson Lehrman Group: Consultancy; Moffitt Cancer Center: Patents & Royalties: field of cellular immunotherapy. Maude: Wugen: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding. Davidson-Moncada: Wugen: Current Employment. Cooper: Wugen: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties; NeoImmune Tech: Patents & Royalties; RiverVest: Consultancy.

Background: CAR-modified T cells targeting CD19 have produced remarkable responses in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL); however, relapse continues to be a substantial challenge. CD19+ relapses, which account for 33-78% of relapses, are associated with loss of CAR T-cell surveillance due to short persistence. Thus, strategies to improve functional persistence to prevent and treat CD19+ relapsed disease are crucial. Here, we report our experience administering reinfusions of murine or humanized 4-1BB CD19 CAR T cells in an effort to prolong persistence in patients with demonstrated short persistence to mitigate relapse risk, treat CD19+ relapsed disease, and produce responses after nonresponse to initial CAR infusion. Methods: This analysis included patients aged Results: Among 229 CAR-naïve and 33 CAR-exposed patients treated with CD19 CAR between 2012-2020, 81 received ≥1 reinfusion (investigational CTL019, n=44; commercial tisagenlecleucel, n=11; huCART19, n=13 CAR-naïve and n=13 CAR-exposed). In addition, 18 patients received PD-1 blockade after their first (n=11) or subsequent (n=7) reinfusions. Indications for first reinfusion were peripheral BCR (CAR-naïve, n=32; CAR-exposed, n=6), hematogones (CAR-naïve, n=21; CAR-exposed, n=4), CD19+ MRD/relapse (CAR-naïve, n=10, CAR-exposed, n=0), and nonresponse to initial infusion (CAR-naïve, n=5, CAR-exposed, n=3). CRS grade ≥2 (Penn scale) occurred in 19 patients (grade 2, n=13; grade 3, n=4; grade 4, n=2). Grade 3-4 events only occurred in patients with active disease at time of reinfusion. Twenty-two patients had an inpatient admission within 30 days of first reinfusion, of which 7 required intensive care unit admission Among the 63 patients reinfused for relapse prevention, 33 (52%) had a CR at day 28. With a median duration of follow-up of 38 mos, 13 experienced a subsequent relapse (7 CD19+, 4 CD19-, 2 CD19-subset negative), 4 received alternative therapy or allogeneic hematopoietic stem cell transplantation (HSCT) in remission, and 16 remain in remission without further therapy at a median of 39 mos after first reinfusion. The median duration of B-cell aplasia was 8 mos (IQR 2-35) after reinfusion. Of the 30 with no response (NR), 10 had a subsequent CD19+ relapse, 15 received alternative therapy or HSCT, and 5 remain in remission without further therapy at a median of 43 mos after reinfusion. CIR and OS were not statistically significantly different between patients with CR or NR (CIR, p=0.26; OS, p=0.25) (Figure A-B). However, at 24 mos after reinfusion, CIR was 29% (95% CI, 11-44%) for CR compared to 61% (95% CI, 24-80%) for NR; OS was 90% (95% CI, 80-100%) for both groups. Of the 10 patients reinfused for relapse, 5 (50%) had a CR; 2 subsequently experienced a CD19+ relapse, 2 received an HSCT in remission, and 1 remains in remission without further therapy at 18 mos after reinfusion. Of the 8 patients reinfused for nonresponse to initial infusion, 7 were evaluable; none had a CR, and all died at a median of 2.5 mos after reinfusion. Conclusions: Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may prolong B-cell aplasia in patients with short CAR persistence and reduce relapse risk, and can induce remissions in patients with CD19+ relapsed disease. Thus, reinfusion may provide an alternative to HSCT for short persistence. However, reinfusion is not effective for patients with nonresponse to initial CAR infusion. Figure 1 Figure 1. Disclosures Callahan: Novartis: Speakers Bureau. Rheingold: Optinose: Other: Spouse's current employment; Pfizer: Research Funding. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: Patents & Royalties; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Maude: Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Wugen: Consultancy.

The impact of immunotherapy has grown exponentially in the past 5 years. Principle illustrations are encouraging results with engineered T cells expressing a chimeric antigen receptor (CAR). This experimental therapy is developing simultaneously in pediatric and adult clinical trials, making this field particularly relevant and exciting for pediatric oncologists.CAR-modified T cells targeting CD19 have produced dramatic antitumor responses in patients with relapsed/refractory B cell acute lymphoblastic leukemia. Clinical trials from several institutions, in both children and adults, using distinct CAR T cell products have demonstrated similar high complete remission rates of 61-93%, with durable remissions observed. Although the development of CARs for other malignancies has lagged behind, research into novel approaches to overcome inherent challenges is promising.Clinical trials of CAR-modified T cells have produced unprecedented results and are anticipated to have a broader impact as this approach expands into other indications, including other cancers and frontline therapy. The potential for long-term disease control, if fully realized, will have a transformative impact on the field.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19- disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy.

125 Background: Prostate specific membrane antigen (PSMA) is a highly expressed tumor-associated antigen potentially amenable to chimeric antigen receptor-modified T (CAR-T) cell therapy for castration-resistant prostate cancer (CRPC). However, a primary challenge to the success of CAR-T therapy in CRPC is the immunosuppressive microenvironment, characterized by high levels of TGFβ. The immunosuppressive functions of TGFβ can be inhibited in T cells using a dominant negative TGFβ receptor (TGFβRdn), thereby enhancing antitumor immunity. Methods: We conducted a first-in-human phase 1 clinical trial to evaluate the feasibility, safety and preliminary efficacy of PSMA-directed/TGFβ-insensitive CAR-T cells (CART-PSMA-TGFβRdn) in patients with metastatic CRPC (NCT03089203). In a 3+3 dose-escalation design, 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 (LD) chemotherapy. In Cohort 3, one patient received 1-3 x 108/m2 CART-PSMA-TGFβRdn cells following a LD chemotherapy regimen of cyclophosphamide and fludarabine (Cy/Flu). In Cohort -3, three patients received 1-3 x 107/m2 CART-PSMA-TGFβRdn cells following Cy/Flu. Patients underwent metastatic tumor biopsies at baseline and on day 10 following treatment. Quantitative PCR of CART-PSMA-TGFβRdn DNA was performed at serial timepoints to evaluate for CAR-T expansion and persistence in peripheral blood and trafficking to target tissues. Multiplex cytokine analysis assessed CART-PSMA-TGFβRdn bioactivity. Results: Ten patients received CART-PSMA-TGFβRdn therapy across dose-level cohorts. All CART-PSMA-TGFβRdn infusion products met target transduction efficiency. Evaluation of CAR-T cellular kinetics demonstrated dose-dependent peripheral blood T cell expansion, as well as tumor tissue trafficking in post-treatment tumor biopsies. At Cohort 2 and above, 5 of 7 treated patients developed grade ≥2 cytokine release syndrome (CRS). Marked increases in inflammatory cytokines (IL-6, IL-15, IL-2, IFNγ) correlated with high-grade CRS events. One grade 5 adverse event (sepsis) occurred in Cohort 3. PSA decline was observed in 6 of 10 patients (median decline -33.2%, range -11.6% to -98.3%), and PSA30 response occurred in 4 of 10 patients (including one patient achieving PSA < 0.1 ng/mL). Conclusions: Adoptive cellular therapy with CART-PSMA-TGFβRdn is safe and feasible in patients with metastatic CRPC. A dose-dependent and lymphodepletion chemotherapy-dependent relationship was observed with CART-PSMA-TGFβRdn cell expansion, cytokine expression, CRS, and anti-tumor effect. Correlative cell trafficking and paired tumor Nanostring analyses will be presented. Future clinical investigations seek to enhance anti-tumor efficacy, while optimizing the therapeutic window. Clinical trial information: NCT03089203.

e22504 Background: Pediatric/YA pts with r/r B-NHL are rare and have heterogenous, aggressive histology and poor prognosis. We report early results for tisagenlecleucel (anti-CD19 CAR-T cell therapy) in pediatric/YA pts with r/r B-NHL. Methods: BIANCA (NCT03610724) is a phase 2, single-arm, global, open-label trial of tisagenlecleucel in pediatric/YA pts with CD19+ r/r B-NHL. Pts must have confirmed mature B-NHL r/r to ≥1 prior lines of therapy and no active CNS involvement. Primary endpoint is ORR. Secondary outcomes include DOR, EFS, safety and pharmacokinetics. Results: As of Nov 4, 2019, 8 pts were enrolled, of whom 4 had large B-cell lymphoma (LBCL), 3 Burkitt lymphoma (BL), and 1 gray zone lymphoma (GZL) (Table). Five pts had ≥2 lines of prior therapy. Suitable apheresis product was harvested in all 8 pts. Five pts were infused and 3 were pending infusion at data cut off. Product was successfully manufactured within specifications for all infused pts. Median time from enrollment to infusion was 33 days (range 30-67). All 5 pts have ≥28 days follow up; 2 pts have ≥3 months follow up (median [range] 85 days [69-97]). All 8 pts received bridging chemotherapy (including 1 pt who also had surgery and 1 who also had radiotherapy). Tisagenlecleucel dose range was 0.3-1.1 × 108 CAR+ viable T cells (weight-based: 0.9-1.7 × 106 CAR+ viable T cells/kg). Cmax (range: Cmax= 8520-14,200 copies/µg; time to Cmax= 2-21 days; n = 4) was within range of expansion observed in pediatric/YA acute lymphoblastic leukemia and adult diffuse LBCL. All 5 pts had CRS; no grade ≥3 CRS was recorded. Three pts had neurologic events, including 2 grade 3/4 events. One pt died due to disease progression. Conclusions: Pediatric/YA pts with r/r B-NHL (including BL) were successfully infused with tisagenlecleucel in the BIANCA trial with a manageable safety profile. Apheresis/manufacturing were feasible in this cohort of rapidly progressing disorders. Tisagenlecleucel was shown to expand in vivo. BIANCA provides the first systematic data on CAR-T cell therapy in highly aggressive, pediatric/YA B-NHL. Planned enrollment is 35 pts (26 infused and evaluable). Clinical trial information: NCT03610724. [Table: see text]

10511 Background: CNS relapse of B-ALL is difficult to treat after cranial radiation or multiple relapses. Durable remissions of relapsed/refractory (r/r) B-ALL have been seen with CD19 CAR T cells; however, most trials excluded patients with active CNS disease. As we observed CAR trafficking into the CSF, we hypothesized that CD19 CAR T cells could control CNS B-ALL. Methods: We identified children and young adults with r/r CNS B-ALL treated on 4 clinical trials of CD19 CAR T cells, CTL019 or CTL119. NCT01626495 and NCT02435849 excluded active CNS disease, while the former in an amendment as well as NCT02374333 and NCT02906371 permitted active CNS disease controlled on therapy. All trials permitted CNS disease that cleared and excluded bulky intracranial disease that did not improve. We analyzed outcomes (CR, RFS) and safety. Results: We identified 65 patients 1-29y (median 10y) with r/r CNS B-ALL (CNS+) of 182 treated with CTL019/CTL119. There were no differences in age, sex, history of SCT or neurologic comorbidities in the CNS+ and CNS- cohorts. CNS+ patients were more likely to be in ≥2nd relapse (74% vs 46%, p < 0.01), to have received cranial radiation (58% vs 11%, p < 0.01), to have detectable CNS disease (p = 0.02) and less bone marrow disease pre-infusion (p < 0.01). At 1 mo post infusion, 62 (95%) CNS+ and 110 (94%) CNS- patients were in CR; 1 in each cohort died of sequelae of CRS and was inevaluable for response. All patients with CNS disease detected pre-infusion cleared by mo 3, including 9 in the CNS+ cohort [5 CNS2 ( < 5 CSF WBC with blasts), 4 CNS3 ( > 5 CSF WBC with blasts or exam/imaging evidence)] and 8 in the CNS- cohort (isolated CNS2 status pre-infusion). There was no difference in RFS (p = 0.28) in the CNS+ and CNS- cohorts [24-mo RFS: 61% (95% CI 46-73%) and 60% (95% CI 48-70%)]. There were 4 CNS relapses in the CNS+ cohort, and 1 in the CNS- cohort. Encephalopathy rate and grade was similar in the CNS+ and CNS- cohorts (52% vs 40% any grade; 12% vs 11% grade 3/4; p = 0.41). There were no deaths due to neurotoxicity (NT) and no statistically significant differences in incidence or severity of any NT or CRS in the CNS+ and CNS- cohorts. Conclusions: The CD19 CAR T cell therapies CTL019/CTL119 are effective at clearing CNS disease and inducing durable remissions in children and young adults with r/r CNS B-ALL. CNS relapse rates are low ( < 3%). Most CD19 CAR T cell trials excluded patients with active CNS disease, primarily due to the risk of NT. We show that patients with r/r CNS B-ALL that is adequately controlled prior to infusion can be safely treated with CD19 CAR T cells, with no increased risk of NT. Clinical trial information: NCT01626495, NCT02435849, NCT02374333, NCT02906371.

CD19-specific chimeric antigen receptor (CAR) T cell therapies have been highly effective against B cell malignancies. We previously demonstrated that differential responses to anti-CD19 CAR T cell therapy in chronic lymphocytic leukemia (CLL) are associated with early memory T cell signature in apheresed, pre-manufacturing T-cells (CAR T-cell precursors). We tested the hypothesis that the composition of CAR-T precursor cells determines clinical efficacy in adult and pediatric Acute Lymphoblastic Leukemia (ALL), Non-Hodgkin's Lymphoma (NHL), Multiple Myeloma (MM), and CLL. Apheresed T cells were engineered to express 4-1BB plus CD3-zeta-signaling CARs targeting CD19, or B cell maturation antigen (BCMA). The same 9-day manufacturing process was used for all trials. CAR T cell kinetics were monitored using a CAR gene-specific quantitative PCR assay and standard clinical response assessments were performed. Apheresed T cells from 36 CLL, 30 adult ALL, 58 pediatric ALL, 33 NHL, and 25 MM patients were immunophenotyped by flow cytometry. The CLL cohort was used to discover phenotypically distinct subpopulations associated with the two main response groups; these associations were validated in the remaining patient cohorts. Eight CD8+ T cell populations or clusters were identified using the shared-nearest-neighbor clustering method (PMID: 31178118) in the CLL cohort. T cell subsets exhibiting naive (cluster 6) or early memory (cluster 4) features were significantly enriched in responding patients, whereas an effector memory CD8 subpopulation (cluster 2) marked the non-responding patients. Mapping these clusters onto apheresed CD8+ T cells from the other four diseases showed that cluster 4 predicted response to CAR T cell therapy in NHL and myeloma but not in adult and pediatric ALL. We also examined the expression of activation-regulated molecules including HLA-DR, Ki67, and exhaustion-related molecules PD1, CTLA4, TIM3, and LAG3. A CD27+ CD8+ population expressing low level CTLA4 but none of the activation or negative regulatory molecules was significantly enriched in responding CLL patients; this cluster validated in NHL and myeloma. A similar analysis on apheresed CD4+ T cells identified an early memory population (cluster 6) enriched in CLL responders, which expresses CCR7 and CD27 but not CD45RO, CD127, CD28, or other late memory/effector molecules. However, this population did not validate in any of the other diseases. Though not statistically significant, the CD4+ clusters with the largest effect size for enrichment in responders from NHL and myeloma trials exhibited early memory T cell features and lack of HLA-DR expression, suggesting that quiescent early memory state in CD4 may also be associated with clinical responses. A separate analysis of checkpoint inhibitory receptors and activation markers in memory CD4 T cell subsets confirmed the early memory, non-activated state of this population in CLL and was validated in myeloma but none of the other diseases. In vivo activation was a shared theme in CD4+ T cells for non-responding patients as well, though these CLL-defined CD4+ apheresed T cells clusters did not significantly validate in other diseases. In summary, our data confirm and extend our predictive biomarker profile in CLL to mature B cell and plasma cell malignancies by showing that a non-cycling, non-activated early memory CD8+ T cell population in pre-manufacturing cells was validated as a biomarker in myeloma, and NHL. We also showed that responder-associated apheresed CD4+ T cells with early memory features identified in CLL after CD19 CAR T infusions are validated in myeloma after BCMA CAR T. Thus, differentiation state and in vivo activation, and potentially exhaustion, separate response groups. Our findings inform next-generation CAR T-cell manufacturing using the populations identified herein as a starting population. Disclosures Pruteanu: Novartis: Employment. Cohen:Poseida Therapeutics, Inc.: Research Funding. Garfall:Surface Oncology: Consultancy; Novartis: Research Funding; Janssen: Research Funding; Amgen: Research Funding; Tmunity: Research Funding. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Gill:Novartis: Research Funding; Tmunity: Research Funding; Carisma: Equity Ownership, Research Funding; Sensei: Consultancy; Aro: Consultancy; Fate: Consultancy. Frey:Novartis: Research Funding. Ruella:Nanostring: Consultancy, Speakers Bureau; Novartis: Patents & Royalties: CART for cancer; AbClon: Membership on an entity's Board of Directors or advisory committees. Lacey:Novartis: Patents & Royalties: Patents related to CAR T cell biomarkers; Tmunity: Research Funding; Novartis: Research Funding. Svoboda:Merck: Research Funding; BMS: Consultancy, Research Funding; Incyte: Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Kite: Consultancy; Seattle Genetics: Consultancy, Research Funding; Kyowa: Consultancy; AstraZeneca: Consultancy. Chong:Tessa: Consultancy; Novartis: Consultancy; Merck: Research Funding. Fraietta:LEK Consulting: Consultancy; Cabaletta: Research Funding; Tmunity: Research Funding. Davis:Cabaletta: Research Funding; Tmunity: Research Funding. Nasta:Rafael: Research Funding; Aileron: Research Funding; Takeda/Millennium: Research Funding; Incyte: Research Funding; Roche/Genentech: Research Funding; Merck: Consultancy; Atara: Research Funding; Debiopharm: Research Funding. Levine:CRC Oncology: Consultancy; Vycellix: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership; Novartis: Consultancy, Patents & Royalties, Research Funding; Cure Genetics: Consultancy; Avectas: Membership on an entity's Board of Directors or advisory committees; Brammer Bio: Membership on an entity's Board of Directors or advisory committees; Incysus: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy. Maude:Kite: Consultancy; Novartis: Consultancy. Schuster:Nordic Nanovector: Honoraria; Pfizer: Honoraria; AstraZeneca: Honoraria; Pharmacyclics: Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Honoraria; Merck: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Novartis: Honoraria, Patents & Royalties: Combination Therapies of CAR and PD-1 Inhibitors with royalties paid to Novartis, Research Funding; AbbVie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding. Stadtmauer:Celgene: Consultancy; Tmunity: Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Research Funding. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Cure Genetics: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards. Porter:Incyte: Membership on an entity's Board of Directors or advisory committees; American Board of Internal Medicine: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Glenmark Pharm: Membership on an entity's Board of Directors or advisory committees; Immunovative: Membership on an entity's Board of Directors or advisory committees; Genentech: Employment; Wiley and Sons: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. June:Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:Novartis: Research Funding, Speakers Bureau; Parker Institute for Cancer Immunotherapy: Research Funding; Stand Up to Cancer: Research Funding; Incyte: Research Funding; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; National Institutes of Health: Research Funding.

We report a patient relapsing 9 months after CD19-targeted CAR T cell (CTL019) infusion with CD19(-) leukemia that aberrantly expressed the anti-CD19 CAR. The CAR gene was unintentionally introduced into a single leukemic B cell during T cell manufacturing, and its product bound in cis to the CD19 epitope on the surface of leukemic cells, masking it from recognition by and conferring resistance to CTL019.

Background & Aim The most common side effect of CD19-directed CAR-T cell therapy is CRS. IL-6 blockade by toci typically results in rapid resolution of severe CRS in CTL019 treated pts; however, the ideal timing of toci remains unclear. Pts with high tumor burden (TB; defined as ≥40% marrow blasts) are at high risk for severe CRS. We hypothesized that preemptive toci administration early in the course of CRS in pts with high TB might decrease acute CRS severity without compromising efficacy. Methods, Results & Conclusion MTD We conducted a two-cohort, open-label study of the timing of toci on CTL019-associated CRS. Pts were 1-24 yo with CD19-expressing relapsed or refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). CTL019 was manufactured at the UPenn Cell and Vaccine Production Facility. Enrolled pts were assigned to high (≥ 40%) or low ( 38.5 C measured twice in 24 hours (“early toci”) and subsequently treated per standard CRS algorithm. Primary endpoint: frequency of gr4 CRS (Penn scale) in ≤5/15 pts. Secondary endpoints: D28 tumor response, remission duration, additional toxicities. RES 78 pts were screened, all were enrolled. 65 pts were evaluable for analysis at data cutoff. 15 pts were assigned to HTBC, 50 pts to LTBC and all were infused with CTL019 (median dose 5.0 × 106 [range 5.6 × 105-3.9 × 107] CAR+ T cells/kg). The overall rate of gr4 CRS was 9% (27% in the HTBC, 4% in the LTBC, Table 1). The overall rate of gr1-4 CRS was 72% (100% in the HTBC, 64% in the LTBC). One pt in the HTBC died from CNS hemorrhage after gr4 CRS. All patients in the HTBC received the early toci intervention. The overall rate of gr3 and 4 CRS was lower than in two prior studies of CTL019 therapy in pediatric r/r B-ALL (18% vs 42% in CHP959 and 48% in ELIANA). The ORR at day 28 follow-up was 97% (87% HTBC, 100% LTBC, with 71% CR and 26% CRi, Table 2). CONC We defined clincially meaningful reduction in severe CRS if ≤5/15 subjects had gr4 CRS in the HTBC; this a priori defined primary end-point was met. Early administration of toci targeted to pts with high TB may reduce the incidence of severe CRS while maintaining excellent efficacy of CTL019 therapy. TB remains a major predictor of toxicity. Longer follow-up is ongoing to assess impact on duration of remission.

Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a recently described subtype of T-ALL characterized by a unique immunophenotype and genomic profile, as well as a high rate of induction failure. Frequent mutations in cytokine receptor and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways led us to hypothesize that ETP-ALL is dependent on JAK/STAT signaling. Here we demonstrate aberrant activation of the JAK/STAT pathway in ETP-ALL blasts relative to non-ETP T-ALL. Moreover, ETP-ALL showed hyperactivation of STAT5 in response to interleukin-7, an effect that was abrogated by the JAK1/2 inhibitor ruxolitinib. In vivo, ruxolitinib displayed activity in 6 of 6 patient-derived murine xenograft models of ETP-ALL, with profound single-agent efficacy in 5 models. Ruxolitinib treatment decreased peripheral blast counts relative to pretreatment levels and compared with control (P < .01) in 5 of 6 ETP-ALL xenografts, with marked reduction in mean splenic blast counts (P < .01) in 6 of 6 samples. Surprisingly, both JAK/STAT pathway activation and ruxolitinib efficacy were independent of the presence of JAK/STAT pathway mutations, raising the possibility that the therapeutic potential of ruxolitinib in ETP-ALL extends beyond those cases with JAK mutations. These findings establish the preclinical in vivo efficacy of ruxolitinib in ETP-ALL, a biologically distinct subtype for which novel therapies are needed.

As a consequence of acquired or intrinsic disease resistance, the prognosis for patients with relapsed or refractory T-cell acute lymphoblastic leukemia (T-ALL) is dismal. Novel, less toxic drugs are clearly needed. One of the most promising emerging therapeutic strategies for cancer treatment is targeted immunotherapy. Immune therapies have improved outcomes for patients with other hematologic malignancies including B-cell ALL; however no immune therapy has been successfully developed for T-ALL. We hypothesize targeting CD38 will be effective against T-ALL. We demonstrate that blasts from patients with T-ALL have robust surface CD38 surface expression and that this expression remains stable after exposure to multiagent chemotherapy. CD38 is expressed at very low levels on normal lymphoid and myeloid cells and on a few tissues of nonhematopoietic origin, suggesting that CD38 may be an ideal target. Daratumumab is a human immunoglobulin G1κ monoclonal antibody that binds CD38, and has been demonstrated to be safe and effective in patients with refractory multiple myeloma. We tested daratumumab in a large panel of T-ALL patient-derived xenografts (PDX) and found striking efficacy in 14 of 15 different PDX. These data suggest that daratumumab is a promising novel therapy for pediatric T-ALL patients.

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.

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.

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.

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 ...

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.