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Supplementary Data from Next-Generation Sequencing of Minimal Residual Disease for Predicting Relapse after Tisagenlecleucel in Children and Young Adults with Acute Lymphoblastic Leukemia

We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection >0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03–9.97; P < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18–10.8; P < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44–7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87–50; P < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33–4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19− (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion.Significance:Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies.See related commentary by Ghorashian and Bartram, p. 2.This article is highlighted in the In This Issue feature, p. 1

RNA and ATACseq data of CART19 cells exposed to BIDko Nalm6

Exposure to death receptor impaired tumor cells results in progressive CART19 dysfunction.

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.

Supplementary Data

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.

Tisagenlecleucel is indicated for pediatric and young adult patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) and adult patients with r/r diffuse large B-cell lymphoma (DLBCL). The tisagenlecleucel chimeric antigen receptor (CAR) contains a murine single-chain variable fragment domain; we examined the effects of humoral and cellular immune responses to tisagenlecleucel on clinical outcomes using 2 validated assays. Data were pooled from the ELIANA (registered at www.clinicaltrials.gov as #NCT02435849) and ENSIGN (#NCT02228096) trials in r/r B-ALL (N = 143) and the JULIET trial (#NCT02445248) in r/r DLBCL (N = 115). Humoral responses were determined by flow cytometric measurement of anti-murine CAR19 (mCAR19) antibodies in serum. Cellular responses were determined using T-cell production of interferon-γ in response to 2 different pools of mCAR19 peptides. Pretreatment anti-mCAR19 antibodies were detected in 81% of patients with r/r B-ALL and 94% of patients with r/r DLBCL. Posttreatment anti-mCAR19 antibodies were higher than patient-specific baseline in 42% of r/r B-ALL and 9% of r/r DLBCL patients. Pretreatment and posttreatment anti-mCAR19 antibodies did not affect tisagenlecleucel cellular kinetics, including maximum concentration and persistence (r2 < 0.05), clinical response (day-28 response, duration of response, and event-free survival), and safety. T-cell responses were consistent over time, with net responses

We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection >0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03–9.97; P < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18–10.8; P < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44–7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87–50; P < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33–4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19− (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion. Significance: Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies. See related commentary by Ghorashian and Bartram, p. 2. This article is highlighted in the In This Issue feature, p. 1

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.

The anti-CD19 chimeric antigen receptor (CAR)–T cell therapy tisagenlecleucel was evaluated in the global, phase 2 JULIET study in adult patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). We correlated tisagenlecleucel cellular kinetics with clinical/product parameters in 111 patients treated in JULIET. Tisagenlecleucel persistence in responders and nonresponders, respectively, was demonstrated for 554 and 400 days maximum by flow cytometry and for 693 and 374 days maximum by quantitative polymerase chain reaction (qPCR). No relationships were identified between cellular kinetics (qPCR) and product characteristics, intrinsic/extrinsic factors, dose, or immunogenicity. Most patients with 3-month response had detectable transgene at time of response and continued persistence for ≥6 months. Expansion (maximal expansion of transgene/CAR-positive T-cell levels in vivo postinfusion [Cmax]) was potentially associated with response duration but this did not reach statistical significance (hazard ratio for a twofold increase in Cmax, 0.79; 95% confidence interval, 0.61-1.01). Tisagenlecleucel expansion was associated with cytokine-release syndrome (CRS) severity and tocilizumab use; no relationships were observed with neurologic events. Transgene levels were associated with B-cell levels. Dose was associated with CRS severity, but this was not statistically significant after adjusting for baseline tumor burden. In contrast to the results from B-cell precursor acute lymphoblastic leukemia (B-ALL) and chronic lymphocytic leukemia, similar exposure was observed in DLBCL in this study regardless of response and expansion was lower in DLBCL than B-ALL, likely from differences in cancer location and/or T-cell intrinsic factors. Relationships between expansion and CRS severity, and lack of relationships between dose and exposure, were similar between DLBCL and B-ALL. Tisagenlecleucel cellular kinetics in adult relapsed/refractory DLBCL improve current understanding of in vivo expansion and its relationships with safety/efficacy endpoints. This trial was registered at www.clinicaltrials.gov as #NCT02445248.

Tisagenlecleucel is a chimeric antigen receptor–T cell therapy that facilitates the killing of CD19+ B cells. A model was developed for the kinetics of tisagenlecleucel and the impact of therapies for treating cytokine release syndrome (tocilizumab and corticosteroids) on expansion. Data from two phase II studies in pediatric and young adult relapsed/refractory B cell acute lymphoblastic leukemia were pooled to evaluate this model and evaluate extrinsic and intrinsic factors that may impact the extent of tisagenlecleucel expansion. The doubling time, initial decline half‐life, and terminal half‐life for tisagenlecleucel were 0.78, 4.3, and 220 days, respectively. No impact of tocilizumab or corticosteroids on the expansion rate was observed. This work represents the first mixed‐effect model‐based analysis of chimeric antigen receptor–T cell therapy and may be clinically impactful as future studies examine prophylactic interventions in patients at risk of higher grade cytokine release syndrome and the effects of these interventions on chimeric antigen receptor–T cell expansion.

Chimeric antigen receptor (CAR)-T cell therapies reprogram T cells to engage and eliminate cancer cells. Patients’ T cells are transduced in vitro using lentiviral or retroviral vectors containing a CAR transgene. Following infusion, CAR-T cells expand in vivo and may persist in the peripheral blood and bone marrow for years. Therefore, monitoring in vivo copies of the CAR transgene requires highly sensitive, validated analytical methods. Herein, we describe the validation of a qPCR assay to detect tisagenlecleucel transgene in patient samples. The limit of detection and lower limit of quantitation were 3.1 and 10 copies/200 ng genomic DNA, respectively, equivalent to ∼50 copies/μg genomic DNA and in alignment with US Food and Drug Administration guidance on bioanalytical method validation. The assay allowed quantitation of the tisagenlecleucel transgene over a wide dynamic range with a high degree of linearity, that is, 101–106 copies/200 ng genomic DNA (R2 ≥ 0.9988). Coefficients of variation of measured transgene copies ranged from 0.2% to 12.8%. A droplet digital PCR assay was performed as a method of validation and showed a strong correlation with the qPCR assay (R2 = 0.9980, p < 0.0001). This qPCR assay is being utilized to monitor tisagenlecleucel expansion and persistence in clinical trials., Graphical Abstract, Highly specific, sensitive, and quantitative analytical assays are needed to monitor the in vivo cellular kinetics of CAR-T cells in the patient after infusion. In this study, Davis and colleagues describe the validation results of a novel qPCR assay designed to specifically identify the tisagenlecleucel CAR transgene in patient samples.

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.

BACKGROUND: In a single-center phase 1-2a study, the anti-CD19 chimeric antigen receptor (CAR) T-cell therapy tisagenlecleucel produced high rates of complete remission and was associated with serious but mainly reversible toxic effects in children and young adults with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL). METHODS: We conducted a phase 2, single-cohort, 25-center, global study of tisagenlecleucel in pediatric and young adult patients with CD19+ relapsed or refractory B-cell ALL. The primary end point was the overall remission rate (the rate of complete remission or complete remission with incomplete hematologic recovery) within 3 months. RESULTS: For this planned analysis, 75 patients received an infusion of tisagenlecleucel and could be evaluated for efficacy. The overall remission rate within 3 months was 81%, with all patients who had a response to treatment found to be negative for minimal residual disease, as assessed by means of flow cytometry. The rates of event-free survival and overall survival were 73% (95% confidence interval [CI], 60 to 82) and 90% (95% CI, 81 to 95), respectively, at 6 months and 50% (95% CI, 35 to 64) and 76% (95% CI, 63 to 86) at 12 months. The median duration of remission was not reached. Persistence of tisagenlecleucel in the blood was observed for as long as 20 months. Grade 3 or 4 adverse events that were suspected to be related to tisagenlecleucel occurred in 73% of patients. The cytokine release syndrome occurred in 77% of patients, 48% of whom received tocilizumab. Neurologic events occurred in 40% of patients and were managed with supportive care, and no cerebral edema was reported. CONCLUSIONS: In this global study of CAR T-cell therapy, a single infusion of tisagenlecleucel provided durable remission with long-term persistence in pediatric and young adult patients with relapsed or refractory B-cell ALL, with transient high-grade toxic effects. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT02435849 .).

Cellular immunotherapy using T cells engineered to express chimeric antigen receptors targeting CD19 (CART19) leads to long-term remission in patients with B-cell malignancies1–5. Unfortunately, a significant fraction of patients demonstrate primary resistance to CART19 or experience relapse after achieving remission. Beyond loss of target antigen, the molecular pathways governing CART19 failure are unknown. Here we demonstrate that death receptors, cell surface signaling molecules that induce target cell apoptosis, are key mediators of leukemic resistance and CART19 failure. Using a functional CRISPR/Cas9-based genome-wide knockout screen6 in B-cell acute lymphoblastic leukemia (ALL), we identified the death receptor signaling pathway as a central regulator of sensitivity to CART19-induced cell death. In the absence of the pro-apoptotic death receptor signaling molecules BID or FADD, ALL cells were resistant to CART19 cytotoxicity, resulting in rapid disease progression in mice. We found that this initial resistance to cytotoxicity led to persistence of tumor cells, which drove the development of T cell dysfunction that further compromised anti-tumor immunity and permitted tumor outgrowth. We validated these findings using clinical samples collected from patients with ALL treated with CD19-targeted CAR T cells and found that expression of pro-apoptotic death receptor pathway genes in pre-treatment tumor samples correlated with CAR T cell expansion and persistence, as well as patient response and overall survival. Our findings indicate that tumor-intrinsic death receptor signaling directly contributes to CAR T cell failure.

Tisagenlecleucel (CTL019) is an investigational immunotherapy that involves reprogramming a patient's own T cells with a transgene encoding a chimeric antigen receptor to identify and eliminate CD19-expressing cells. We previously reported that CTL019 achieved impressive clinical efficacy in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), including the expansion and persistence of CTL019 cells, which correlates with response to therapy. Here, we performed formal cellular kinetic analyses of CTL019 in a larger cohort of 103 patients treated with CTL019 in 2 different diseases (ALL and CLL). CTL019 was measured in peripheral blood and bone marrow, using quantitative polymerase chain reaction and flow cytometry. CTL019 levels in peripheral blood typically peaked at 10 to 14 days postinfusion and then declined slowly over time. Patients with complete response (CR)/CR with incomplete count recovery had higher levels of CTL019 in peripheral blood, with greater maximal concentration and area under the curve values compared with nonresponding patients (P < .0001 for each). CTL019 transgene levels were measurable up to 780 days in peripheral blood. CTL019 trafficking and persistence were observed in bone marrow and cerebrospinal fluid. CTL019 expansion correlated with severity of cytokine release syndrome (CRS) and preinfusion tumor burden in pediatric ALL. The results described here are the first detailed formal presentation of cellular kinetics across 2 diseases and highlight the importance of the application of in vivo cellular kinetic analyses to characterize clinical efficacy and CRS severity associated with CTL019 therapy.

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 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 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.

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 ...

e15056Background: Analyses supporting a safe and efficacious dose for CAR therapies are not well defined and vary across CAR-T programs. These analyses present key considerations to support tisagen...

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.