Publications by authors named "Stephan A Grupp"

175 Publications

Cytosine Base Editing Enables Quadruple-Edited Allogeneic CAR-T Cells for T-ALL.

Blood 2022 May 13. Epub 2022 May 13.

The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, United States.

Allogeneic chimeric antigen receptor T cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CART have been created using gene editing techniques that induce DNA double-stranded breaks (DSBs), resulting in unintended on-target editing outcomes with potentially unforeseen consequences. Cytosine base editors (CBEs) install C•G to T•A point mutations in T cells with between 90-99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared to CRISPR-Cas9. Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using four simultaneous base edits. We show that CBE, unlike CRISPR-Cas9, does not impact T-cell proliferation, lead to aberrant DNA damage response pathway activation or result in karyotypic abnormalities following multiplexed editing. We demonstrate 7CAR8 to be highly efficacious against T-cell acute lymphoblastic leukemia (T-ALL) using multiple in vitro and in vivo models. Thus, CBE is a promising technology for applications requiring multiplexed gene editing and can be used to manufacture quadruple-edited 7CAR8 cells with high potential for clinical translation for relapsed and refractory T-ALL.
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http://dx.doi.org/10.1182/blood.2022015825DOI Listing
May 2022

Preinfusion factors impacting relapse immunophenotype following CD19 CAR T cells.

Blood Adv 2022 Apr 28. Epub 2022 Apr 28.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health (NIH), Bethesda, Maryland, United States.

Relapse following chimeric antigen receptor (CAR) T-cell therapy directed against CD19 for relapsed/refractory B-acute lymphoblastic leukemia (r/r B-ALL) remains a significant challenge. Three main patterns of relapse predominate: CD19 positive (CD19pos) relapse, CD19 negative (CD19neg) relapse, and lineage switch (LS). Development and validation of risk factors that predict relapse phenotype could help define potential pre- or post-CAR T-cell infusion interventions aimed at decreasing relapse. Our group sought to extensively characterize pre-infusion risk factors associated with the development of each relapse pattern via a multicenter, retrospective review of children and young adults with r/r B-ALL treated with a murine-based CD19-CAR construct. Of 420 CAR-treated patients, 166 (39.5%) relapsed, including 83 (50%) CD19pos, 68 (41%) CD19neg, and 12 (7.2%) LS relapses. A greater cumulative number of prior complete remissions was associated with CD19pos relapses, whereas high pre-infusion disease burden, prior blinatumomab non-response, older age, and 4-1BB CAR construct were associated with CD19neg relapses. The presence of a KMT2A rearrangement was the only pre-infusion risk factor associated with LS. The median overall survival following a post-CAR relapse was 11.9 months (95% CI 9-17 months) and was particularly dismal in patients experiencing a LS, with no long-term survivors following this pattern of relapse. Given the poor outcomes for those with post-CAR relapse, study of relapse prevention strategies, such as consolidative hematopoietic stem cell transplant, is critical and warrants further investigation on prospective clinical trials.
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http://dx.doi.org/10.1182/bloodadvances.2022007423DOI Listing
April 2022

Tisagenlecleucel in pediatric and young adult patients with Down syndrome-associated relapsed/refractory acute lymphoblastic leukemia.

Leukemia 2022 Apr 14. Epub 2022 Apr 14.

Department of Pediatrics and Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, and the Division of Oncology, Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.

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.
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http://dx.doi.org/10.1038/s41375-022-01550-zDOI Listing
April 2022

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

Blood Cancer Discov 2022 01 1;3(1):66-81. Epub 2021 Dec 1.

Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

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 ( = 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; < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18-10.8; < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44-7.69; = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87-50; < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33-4.79; = 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...
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http://dx.doi.org/10.1158/2643-3230.BCD-21-0095DOI Listing
January 2022

Potential Role of IFNγ Inhibition in Refractory Cytokine Release Syndrome Associated with CAR T-cell Therapy.

Blood Cancer Discov 2022 Mar;3(2):90-94

Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Summary: Here we review the pathophysiology and management of cytokine release syndrome (CRS) secondary to immunotherapy, and potential options for CRS refractory to IL6 inhibition and glucocorticoids, for which there are no proven treatments. To illustrate, we describe a patient with B-cell acute lymphoblastic leukemia who developed refractory grade 4 CRS following CD19-directed chimeric antigen receptor T-cell therapy, treated with tocilizumab, methylprednisolone, siltuximab, and the IFNγ inhibitor emapalumab, with complete remission from leukemia for 12 months. See related article by Bailey et al., p. 136 (15).
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http://dx.doi.org/10.1158/2643-3230.BCD-21-0203DOI Listing
March 2022

Unrelated donor α/β T cell- and B cell-depleted HSCT for the treatment of pediatric acute leukemia.

Blood Adv 2022 Feb;6(4):1175-1185

Department of Pediatrics, Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA.

Unrelated donor (URD) hematopoietic stem cell transplant (HSCT) is associated with an increased risk of severe graft-versus-host disease (GVHD). TCRαβ/CD19 depletion may reduce this risk, whereas maintaining graft-versus-leukemia. Outcome data with TCRαβ/CD19 depletion generally describe haploidentical donors, with relatively few URDs. We hypothesized that TCRαβ/CD19-depletion would attenuate the risks of GVHD and relapse for URD HSCT. Sixty pediatric and young adult (YA) patients with hematologic malignancies who lacked a matched-related donor were enrolled at 2 large pediatric transplantation centers between October 2014 and September 2019. All patients with acute leukemia had minimal residual disease testing, and DP typing was available for 77%. All patients received myeloablative total body irradiation- or busulfan-based conditioning with no posttransplant immune suppression. Engraftment occurred in 98%. Four-year overall survival was 69% (95% confidence interval [CI], 52%-81%), and leukemia-free survival was 64% (95% CI, 48%-76%), with no difference between lymphoid and myeloid malignancies (P = .6297 and P = .5441, respectively). One patient (1.7%) experienced primary graft failure. Relapse occurred in 11 patients (3-year cumulative incidence, 21%; 95% CI, 11-34), and 8 patients (cumulative incidence, 15%; 95% CI, 6.7-26) experienced nonrelapse mortality. Grade III to IV acute GVHD was seen in 8 patients (13%), and 14 patients (26%) developed chronic GVHD, of which 6 (11%) had extensive disease. Nonpermissive DP mismatch was associated with higher likelihood of acute GVHD (odds ratio, 16.50; 95% CI, 1.67-163.42; P = .0166) but not with the development of chronic GVHD. URD TCRαβ/CD19-depleted peripheral HSCT is a safe and effective approach to transplantation for children/YAs with leukemia. This trial was registered at www.clinicaltrials.gov as #NCT02323867.
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http://dx.doi.org/10.1182/bloodadvances.2021005492DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8864664PMC
February 2022

Impact of high-risk cytogenetics on outcomes for children and young adults receiving CD19-directed CAR T-cell therapy.

Blood 2022 Apr;139(14):2173-2185

Division of Oncology and Cancer Immunotherapy Program, Children's Hospital of Philadelphia, Philadelphia, PA.

Chimeric antigen receptor (CAR) T-cell therapy can induce durable remissions of relapsed/refractory B-acute lymphoblastic leukemia (ALL). However, case reports suggested differential outcomes mediated by leukemia cytogenetics. We identified children and young adults with relapsed/refractory CD19+ ALL/lymphoblastic lymphoma treated on 5 CD19-directed CAR T-cell (CTL019 or humanized CART19) clinical trials or with commercial tisagenlecleucel from April 2012 to April 2019. Patients were hierarchically categorized according to leukemia cytogenetics: High-risk lesions were defined as KMT2A (MLL) rearrangements, Philadelphia chromosome (Ph+), Ph-like, hypodiploidy, or TCF3/HLF; favorable as hyperdiploidy or ETV6/RUNX1; and intermediate as iAMP21, IKZF1 deletion, or TCF3/PBX1. Of 231 patients aged 1 to 29, 74 (32%) were categorized as high risk, 28 (12%) as intermediate, 43 (19%) as favorable, and 86 (37%) as uninformative. Overall complete remission rate was 94%, with no difference between strata. There was no difference in relapse-free survival (RFS; P = .8112), with 2-year RFS for the high-risk group of 63% (95% confidence interval [CI], 52-77). There was similarly no difference seen in overall survival (OS) (P = .5488), with 2-year OS for the high-risk group of 70% (95% CI, 60-82). For patients with KMT2A-rearranged infant ALL (n = 13), 2-year RFS was 67% (95% CI, 45-99), and OS was 62% (95% CI, 40-95), with multivariable analysis demonstrating no increased risk of relapse (hazard ratio, 0.70; 95% CI, 0.21-2.90; P = .7040) but a higher proportion of relapses associated with myeloid lineage switch and a 3.6-fold increased risk of all-cause death (95% CI, 1.04-12.75; P = .0434). CTL019/huCART19/tisagenlecleucel are effective at achieving durable remissions across cytogenetic categories. Relapsed/refractory patients with high-risk cytogenetics, including KMT2A-rearranged infant ALL, demonstrated high RFS and OS probabilities at 2 years.
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http://dx.doi.org/10.1182/blood.2021012727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8990372PMC
April 2022

Blinatumomab Nonresponse and High-Disease Burden Are Associated With Inferior Outcomes After CD19-CAR for B-ALL.

J Clin Oncol 2022 03 12;40(9):932-944. Epub 2021 Nov 12.

National Cancer Institute/Center for Cancer Research, Pediatric Oncology Branch, National Institutes of Health, Bethesda, MD.

Purpose: CD19-targeted chimeric antigen receptor T cells (CD19-CAR) and blinatumomab effectively induce remission in relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) but are also associated with CD19 antigen modulation. There are limited data regarding the impact of prior blinatumomab exposure on subsequent CD19-CAR outcomes.

Patients And Methods: We conducted a multicenter, retrospective review of children and young adults with relapsed or refractory ALL who received CD19-CAR between 2012 and 2019. Primary objectives addressed 6-month relapse-free survival (RFS) and event-free survival (EFS), stratified by blinatumomab use. Secondary objectives included comparison of longer-term survival outcomes, complete remission rates, CD19 modulation, and identification of factors associated with EFS.

Results: Of 420 patients (median age, 12.7 years; interquartile range, 7.1-17.5) treated with commercial tisagenlecleucel or one of three investigational CD19-CAR constructs, 77 (18.3%) received prior blinatumomab. Blinatumomab-exposed patients more frequently harbored rearrangements and underwent a prior stem-cell transplant than blinatumomab-naïve patients. Among patients evaluable for CD19-CAR response (n = 412), blinatumomab nonresponders had lower complete remission rates to CD19-CAR (20 of 31, 64.5%) than blinatumomab responders (39 of 42, 92.9%) or blinatumomab-naive patients (317 of 339, 93.5%), < .0001. Following CD19-CAR, blinatumomab nonresponders had worse 6-month EFS (27.3%; 95% CI, 13.6 to 43.0) compared with blinatumomab responders (66.9%; 95% CI, 50.6 to 78.9; < .0001) or blinatumomab-naïve patients (72.6%; 95% CI, 67.5 to 77; < .0001) and worse RFS. High-disease burden independently associated with inferior EFS. CD19-dim or partial expression (preinfusion) was more frequently seen in blinatumomab-exposed patients (13.3% 6.5%; = .06) and associated with lower EFS and RFS.

Conclusion: With the largest series to date in pediatric CD19-CAR, and, to our knowledge, the first to study the impact of sequential CD19 targeting, we demonstrate that blinatumomab nonresponse and high-disease burden were independently associated with worse RFS and EFS, identifying important indicators of long-term outcomes following CD19-CAR.
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http://dx.doi.org/10.1200/JCO.21.01405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8937010PMC
March 2022

CD19-targeted chimeric antigen receptor T-cell therapy for CNS relapsed or refractory acute lymphocytic leukaemia: a post-hoc analysis of pooled data from five clinical trials.

Lancet Haematol 2021 Oct;8(10):e711-e722

Division of Oncology and Cancer Immunotherapy Program, Children's Hospital of Philadelphia, Philadelphia, PA, USA; Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA; Center for Cellular Immunotherapies, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. Electronic address:

Background: CNS relapse of acute lymphocytic leukaemia is difficult to treat. Durable remissions of relapsed or refractory B-cell acute lymphocytic leukaemia have been observed following treatment with CD19-directed chimeric antigen receptor (CAR) T cells; however, most trials have excluded patients with active CNS disease. We aimed to assess the safety and activity of CAR T-cell therapy in patients with a history of CNS relapsed or refractory B-cell acute lymphocytic leukaemia.

Methods: In this post-hoc analysis, we included 195 patients (aged 1-29 years; 110 [56%] male and 85 [44%] female) with relapsed or refractory CD19-positive acute lymphocytic leukaemia or lymphocytic lymphoma from five clinical trials (Pedi CART19, 13BT022, ENSIGN, ELIANA, and 16CT022) done at the Children's Hospital of Philadelphia (Philadelphia, PA, USA), in which participants received CD19-directed CAR T-cell therapy between April 17, 2012, and April 16, 2019. The trials required control of CNS disease at enrolment and infusion and excluded treatment in the setting of acute neurological toxic effects (>grade 1 in severity) or parenchymal lesions deemed to increase the risk of neurotoxicity. 154 patients from Pedi CART19, ELIANA, ENSIGN, and 16CT022 received tisagenlecleucel and 41 patients from the 13BT022 trial received the humanised CD19-directed CAR, huCART19. We categorised patients into two strata on the basis of CNS status at relapse or within the 12 months preceding CAR T-cell infusion-either CNS-positive or CNS-negative disease. Patients with CNS-positive disease were further divided on the basis of morphological bone marrow involvement-either combined bone marrow and CNS involvement, or isolated CNS involvement. Endpoints were the proportion of patients with complete response at 28 days after infusion, Kaplan-Meier analysis of relapse-free survival and overall survival, and the incidence of cytokine release syndrome and neurotoxicity.

Findings: Of all 195 patients, 66 (34%) were categorised as having CNS-positive disease and 129 (66%) as having CNS-negative disease, and 43 (22%) were categorised as having isolated CNS involvement. The median length of follow-up was 39 months (IQR 25-49) in the CNS-positive stratum and 36 months (18-49) in the CNS-negative stratum. The proportion of patients in the CNS-positive stratum with a complete response at 28 days after infusion was similar to that in the CNS-negative stratum (64 [97%] of 66 vs 121 [94%] of 129; p=0·74), with no significant difference in relapse-free survival (60% [95% CI 49-74] vs 60% [51-71]; p=0·50) or overall survival (83% [75-93] vs 71% [64-79]; p=0·39) at 2 years between the two groups. Overall survival at 2 years was significantly higher in patients with isolated CNS involvement compared with those with bone marrow involvement (91% [82-100] vs 71% [64-78]; p=0·046). The incidence and severity of neurotoxicity (any grade, 53 [41%] vs 38 [58%]; grade 1, 24 [19%] vs 20 [30%]; grade 2, 14 [11%] vs 10 [15%]; grade 3, 12 [9%] vs 6 [9%], and grade 4, 3 [2%] vs 2 [3%]; p=0·20) and cytokine release syndrome (any grade, 110 [85%] vs 53 [80%]; grade 1, 12 [9%] vs 2 [3%]; grade 2, 61 [47%] vs 38 [58%]; grade 3, 18 [14%] vs 7 [11%] and grade 4, 19 [15%] vs 6 [9%]; p=0·26) did not differ between the CNS-negative and the CNS-positive disease strata.

Interpretation: Tisagenlecleucel and huCART19 are active at clearing CNS disease and maintaining durable remissions in children and young adults with CNS relapsed or refractory B-cell acute lymphocytic leukaemia or lymphocytic lymphoma, without increasing the risk of severe neurotoxicity; although care should be taken in the timing of therapy and disease control to mitigate this risk. These preliminary findings support the use of these CAR T-cell therapies for patients with CNS relapsed or refractory B-cell acute lymphocytic leukaemia.

Funding: Children's Hospital of Philadelphia Frontier Program.
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http://dx.doi.org/10.1016/S2352-3026(21)00238-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9026766PMC
October 2021

Distinct Bioenergetic Features of Human Invariant Natural Killer T Cells Enable Retained Functions in Nutrient-Deprived States.

Front Immunol 2021 9;12:700374. Epub 2021 Aug 9.

Division of Infectious Diseases, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, United States.

Invariant natural killer T (iNKT) cells comprise a unique subset of lymphocytes that are primed for activation and possess innate NK-like functional features. Currently, iNKT cell-based immunotherapies remain in early clinical stages, and little is known about the ability of these cells to survive and retain effector functions within the solid tumor microenvironment (TME) long-term. In conventional T cells (T), cellular metabolism is linked to effector functions and their ability to adapt to the nutrient-poor TME. In contrast, the bioenergetic requirements of iNKT cells - particularly those of human iNKT cells - at baseline and upon stimulation are not well understood; neither is how these requirements affect effector functions such as production of cytokines and cytolytic proteins. We find that unlike T, human iNKT cells are not dependent upon glucose or glutamine for these effector functions upon stimulation with anti-CD3 and anti-CD28. Additionally, transcriptional profiling revealed that stimulated human iNKT cells are less glycolytic than T and display higher expression of fatty acid oxidation (FAO) and adenosine monophosphate-activated protein kinase (AMPK) pathway genes. Furthermore, stimulated iNKT cells displayed higher mitochondrial mass and membrane potential relative to T. Real-time Seahorse metabolic flux analysis revealed that stimulated human iNKT cells utilize fatty acids as substrates for oxidation more than stimulated T Together, our data suggest that human iNKT cells possess different bioenergetic requirements from T and display a more oxidative metabolic program relative to effector T. Importantly, iNKT cell-based immunotherapeutic strategies could co-opt such unique features of iNKT cells to improve their efficacy and longevity of anti-tumor responses.
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http://dx.doi.org/10.3389/fimmu.2021.700374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380770PMC
December 2021

Tisagenlecleucel immunogenicity in relapsed/refractory acute lymphoblastic leukemia and diffuse large B-cell lymphoma.

Blood Adv 2021 12;5(23):4980-4991

Department of Pediatrics, Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX; and.

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 <1% at baseline and posttreatment time points in a majority of patients and no effect on transgene expansion or persistence or outcomes. Presence of baseline and/or posttreatment anti-mCAR19 antibodies or T-cell responses did not alter the activity of tisagenlecleucel in patients with r/r B-ALL or r/r DLBCL.
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http://dx.doi.org/10.1182/bloodadvances.2020003844DOI Listing
December 2021

Pooled safety analysis of tisagenlecleucel in children and young adults with B cell acute lymphoblastic leukemia.

J Immunother Cancer 2021 08;9(8)

Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Background: Tisagenlecleucel, an anti-CD19 chimeric antigen receptor T cell therapy, has demonstrated efficacy in children and young adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) in two multicenter phase 2 trials (ClinicalTrials.gov, NCT02435849 (ELIANA) and NCT02228096 (ENSIGN)), leading to commercialization of tisagenlecleucel for the treatment of patients up to age 25 years with B-ALL that is refractory or in second or greater relapse.

Methods: A pooled analysis of 137 patients from these trials (ELIANA: n=79; ENSIGN: n=58) was performed to provide a comprehensive safety profile for tisagenlecleucel.

Results: Grade 3/4 tisagenlecleucel-related adverse events (AEs) were reported in 77% of patients. Specific AEs of interest that occurred ≤8 weeks postinfusion included cytokine-release syndrome (CRS; 79% (grade 4: 22%)), infections (42%; grade 3/4: 19%), prolonged (not resolved by day 28) cytopenias (40%; grade 3/4: 34%), neurologic events (36%; grade 3: 10%; no grade 4 events), and tumor lysis syndrome (4%; all grade 3). Treatment for CRS included tocilizumab (40%) and corticosteroids (23%). The frequency of neurologic events increased with CRS severity (p<0.001). Median time to resolution of grade 3/4 cytopenias to grade ≤2 was 2.0 (95% CI 1.87 to 2.23) months for neutropenia, 2.4 (95% CI 1.97 to 3.68) months for lymphopenia, 2.0 (95% CI 1.87 to 2.27) months for leukopenia, 1.9 (95% CI 1.74 to 2.10) months for thrombocytopenia, and 1.0 (95% CI 0.95 to 1.87) month for anemia. All patients who achieved complete remission (CR)/CR with incomplete hematologic recovery experienced B cell aplasia; however, as nearly all responders also received immunoglobulin replacement, few grade 3/4 infections occurred >1 year postinfusion.

Conclusions: This pooled analysis provides a detailed safety profile for tisagenlecleucel during the course of clinical trials, and AE management guidance, with a longer follow-up duration compared with previous reports.
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http://dx.doi.org/10.1136/jitc-2020-002287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8344270PMC
August 2021

Humanized CD19-Targeted Chimeric Antigen Receptor (CAR) T Cells in CAR-Naive and CAR-Exposed Children and Young Adults With Relapsed or Refractory Acute Lymphoblastic Leukemia.

J Clin Oncol 2021 09 22;39(27):3044-3055. Epub 2021 Jun 22.

Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA.

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.
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http://dx.doi.org/10.1200/JCO.20.03458DOI Listing
September 2021

CAR-T cells: Early successes in blood cancer and challenges in solid tumors.

Acta Pharm Sin B 2021 May 2;11(5):1129-1147. Epub 2020 Nov 2.

Department of Chemical Engineering, Northeastern University, Boston, MA 02115, USA.

New approaches to cancer immunotherapy have been developed, showing the ability to harness the immune system to treat and eliminate cancer. For many solid tumors, therapy with checkpoint inhibitors has shown promise. For hematologic malignancies, adoptive and engineered cell therapies are being widely developed, using cells such as T lymphocytes, as well as natural killer (NK) cells, dendritic cells, and potentially others. Among these adoptive cell therapies, the most active and advanced therapy involves chimeric antigen receptor (CAR)-T cells, which are T cells in which a chimeric antigen receptor is used to redirect specificity and allow T cell recognition, activation and killing of cancers, such as leukemia and lymphoma. Two autologous CAR-T products have been approved by several health authorities, starting with the U.S. Food and Drug Administration (FDA) in 2017. These products have shown powerful, inducing, long-lasting effects against B cell cancers in many cases. In distinction to the results seen in hematologic malignancies, the field of using CAR-T products against solid tumors is in its infancy. Targeting solid tumors and trafficking CAR-T cells into an immunosuppressive microenvironment are both significant challenges. The goal of this review is to summarize some of the most recent aspects of CAR-T cell design and manufacturing that have led to successes in hematological malignancies, allowing the reader to appreciate the barriers that must be overcome to extend CAR-T therapies to solid tumors successfully.
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http://dx.doi.org/10.1016/j.apsb.2020.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8144892PMC
May 2021

Single-cell multiomics dissection of basal and antigen-specific activation states of CD19-targeted CAR T cells.

J Immunother Cancer 2021 05;9(5)

Department of Biomedical Engineering, Yale University, New Haven, Connecticut, USA

Background: Autologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 molecule have transformed the therapeutic landscape in patients with highly refractory leukemia and lymphoma, and the use of donor-generated allogeneic CAR T is paving the way for further breakthroughs in the treatment of cancer. However, it remains unknown how the intrinsic heterogeneities of these engineered cells mediate therapeutic efficacy and whether allogeneic products match the effectiveness of autologous therapies.

Methods: Using single-cell mRNA sequencing in conjunction with CITE-seq, we performed multiomics characterization of CAR T cells generated from healthy donor and patients with acute lymphoblastic leukemia. CAR T cells used in this study were manufactured at the University of Pennsylvania through lentiviral transduction with a CD19-4-1BB-CD3ζ construct. Besides the baseline condition, we engineered NIH-3T3 cells with human CD19 or mesothelin expression to conduct ex vivo antigen-specific or non-antigen stimulation of CAR T cells through 6-hour coculture at a 1:1 ratio.

Results: We delineated the global cellular and molecular CAR T landscape and identified that transcriptional CAR tonic signaling was regulated by a mixture of early activation, exhaustion signatures, and cytotoxic activities. On CD19 stimulation, we illuminated the disparities of CAR T cells derived from different origins and found that donor CAR T had more pronounced activation level in correlation with the upregulation of major histocompatibility complex class II genes compared with patient CAR T cells. This finding was independently validated in additional datasets from literature. Furthermore, GM-CSF() expression was found to be associated with functional gene productions, but it induced little impact on the CAR T activation.

Conclusions: Through integrated multiomics profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the transcriptional, phenotypic, functional, and metabolic profiles of donor and patient CAR T cells, providing mechanistic basis for ameliorating clinical outcomes and developing next-generation 'off- the-shelf' allogeneic products.
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http://dx.doi.org/10.1136/jitc-2020-002328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8137188PMC
May 2021

Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells.

Nat Med 2021 05 22;27(5):842-850. Epub 2021 Apr 22.

Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

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.
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http://dx.doi.org/10.1038/s41591-021-01326-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8451032PMC
May 2021

Absolute lymphocyte count proliferation kinetics after CAR T-cell infusion impact response and relapse.

Blood Adv 2021 04;5(8):2128-2136

Division of Hematopathology, The Children's Hospital of Philadelphia, Philadelphia, PA.

CD19-directed chimeric antigen receptor (CAR) T cells show characteristic proliferation kinetics after infusion that correlate with response. Clearance of circulating disease, B-cell aplasia (BCA), and cytokine release syndrome (CRS) are used to observe CAR T-cell function, given the lack of commercial CAR T-cell measurement assays. We investigated the utility of common hematology laboratory parameters in 166 patients with B-cell acute lymphoblastic leukemia (B-ALL) who were treated with CAR T-cell therapy targeting CD19. CAR T-cell infusion was followed by disappearance of circulating blasts in 86% of patients at a median of 6 days. After a lag phase, there was a rapid expansion in absolute lymphocyte count (ALC) in the second week that coincided with the appearance of atypical lymphocytes. The expansion phase was followed by a contraction phase with a concomitant decrease in atypical lymphocytes. In vitro CAR T-cell studies showed similar kinetics and morphological changes. Peak ALC and overall expansion was greater in sustained responders compared with that in nonresponders. Patients with early loss of BCA and those with eventual CD19+ minimal residual disease/relapse showed lower overall lymphocyte expansion compared with the controls. Pleomorphic lymphocytosis was noted in the cerebrospinal fluid at post-CAR time points. We conclude that lymphocyte counts and differential can also be used to evaluate CAR T-cell expansion after infusion, along with BCA and CRS. This is the first report to characterize the morphology of CAR T cells and determine the utility of lymphocyte kinetics.
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http://dx.doi.org/10.1182/bloodadvances.2020004038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095130PMC
April 2021

Immune Reconstitution Following TCRαβ/CD19-Depleted Hematopoietic Cell Transplantation for Hematologic Malignancy in Pediatric Patients.

Transplant Cell Ther 2021 02 10;27(2):169.e1-169.e9. Epub 2020 Dec 10.

Division of Oncology, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania. Electronic address:

TCRαβ/CD19-depleted HCT has been used with excellent outcomes in pediatric patients with hematologic malignancies, and several studies have demonstrated rapid immune reconstitution in the nonmalignant setting. However, immune recovery following TCRαβ/CD19-depleted hematopoietic cell transplantation (HCT) for malignancy remains incompletely elucidated. Furthermore, the majority of studies to date have used haploidentical and matched unrelated donors. Here we report results of immune reconstitution following TCRαβ/CD19-depleted HCT for hematologic malignancy in 51 pediatric patients with hematologic malignancies, the majority of whom received grafts from unrelated donors. Grafts were from matched unrelated (n = 20), mismatched unrelated (n = 20), and haploidentical (n = 11) donors. The median CD34 cell dose was 10.2 × 10/kg (range, 4.54 to 20 × 10/kg), and the median TCRαβ cell dose was 2.53 × 10/kg (range, 0 to 44.9 × 10/kg). Conditioning was myeloablative with either busulfan or total body irradiation, cyclophosphamide, and thiotepa. Thirty-three patients also received rabbit antithymocyte globulin. No prophylactic post-transplantation immune suppression was routinely given. Forty-three patients received rituximab on day +1 for recipient positive Epstein-Barr virus serology. Forty-nine patients (96%) engrafted with a median time to neutrophil recovery of 13 days (range, 8 to 30 days). Thirty-seven patients (73%) are alive at a median follow-up of 25 months (range, 6 to 50 months). Nine patients (18%) developed grade II-IV acute graft-versus-host disease (GVHD), and 5 patients (11%) developed extensive chronic GVHD. Twenty-six patients (51%) experienced viral reactivation. T cell reconstitution was rapid with significant numbers of CD3, CD4, and CD8 T cells present on first assessment at 4 months post-HCT, and significant numbers of naïve CD4 T cells were present by 8 months post-HCT. Chronic GVHD was associated with delayed T cell recovery; however, T cell reconstitution was not affected by underlying diagnosis, donor source, TCRαβ T cell dose, conditioning regimen, or use of antithymocyte globulin. B cell recovery mirrored T cell recovery, and i.v. Ig was discontinued at a median of 8 months (range, 4 to 22 months) post-HCT in patients alive and relapse-free at last follow-up. Immune reconstitution is rapid following TCRαβ/CD19-depleted HCT in pediatric patients with hematologic malignancies. Donor graft source, haploidentical or unrelated, did not affect immune reconstitution. Viral reactivation is common in the first 100 days post-HCT, indicating that improved T cell defense is needed in the early post-HCT period.
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http://dx.doi.org/10.1016/j.jtct.2020.10.006DOI Listing
February 2021

Integrative Bulk and Single-Cell Profiling of Premanufacture T-cell Populations Reveals Factors Mediating Long-Term Persistence of CAR T-cell Therapy.

Cancer Discov 2021 09 5;11(9):2186-2199. Epub 2021 Apr 5.

Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

The adoptive transfer of chimeric antigen receptor (CAR) T cells represents a breakthrough in clinical oncology, yet both between- and within-patient differences in autologously derived T cells are a major contributor to therapy failure. To interrogate the molecular determinants of clinical CAR T-cell persistence, we extensively characterized the premanufacture T cells of 71 patients with B-cell malignancies on trial to receive anti-CD19 CAR T-cell therapy. We performed RNA-sequencing analysis on sorted T-cell subsets from all 71 patients, followed by paired Cellular Indexing of Transcriptomes and Epitopes (CITE) sequencing and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) on T cells from six of these patients. We found that chronic IFN signaling regulated by was associated with poor CAR T-cell persistence across T-cell subsets, and that the regulon not only associates with the favorable naïve T-cell state, but is maintained in effector T cells among patients with long-term CAR T-cell persistence. These findings provide key insights into the underlying molecular determinants of clinical CAR T-cell function. SIGNIFICANCE: To improve clinical outcomes for CAR T-cell therapy, there is a need to understand the molecular determinants of CAR T-cell persistence. These data represent the largest clinically annotated molecular atlas in CAR T-cell therapy to date, and significantly advance our understanding of the mechanisms underlying therapeutic efficacy..
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http://dx.doi.org/10.1158/2159-8290.CD-20-1677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419030PMC
September 2021

Improving and Maintaining Responses in Pediatric B-Cell Acute Lymphoblastic Leukemia Chimeric Antigen Receptor-T Cell Therapy.

Cancer J 2021 Mar-Apr 01;27(2):151-158

Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA.

Abstract: Chimeric antigen receptor T therapy has heralded a new era in the treatment of acute lymphoblastic leukemia (ALL) and other hematologic malignancies. In this autologous immunotherapy, patient-derived T cells are genetically engineered and then infused back to kill the leukemia cells. The observed response rates in ALL are a testament to the success of this therapy. However, there have been instances where the patients either did not respond or relapsed after initial response. Emergence of resistance due to antigen loss and T-cell exhaustion has been observed. This poses a challenge in making this therapy successful for every ALL patient and warrants deeper understanding of emergence of resistance and potential approaches to overcome them. Here we discuss current perspectives and advances in this area.
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http://dx.doi.org/10.1097/PPO.0000000000000513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130572PMC
September 2021

Will allogeneic CAR T cells for CD19 malignancies take autologous CAR T cells 'off the shelf'?

Nat Rev Clin Oncol 2021 04;18(4):195-196

Cancer Immunotherapy Program, Cellular Therapy and Transplant Section, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

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http://dx.doi.org/10.1038/s41571-021-00485-1DOI Listing
April 2021

Practical guidelines for monitoring and management of coagulopathy following tisagenlecleucel CAR T-cell therapy.

Blood Adv 2021 01;5(2):593-601

Department of Pediatric Hematology-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium.

Cytokine release syndrome (CRS) is a systemic inflammatory response associated with chimeric antigen receptor T-cell (CAR-T) therapies. In severe cases, CRS can be associated with coagulopathy and hypofibrinogenemia. We present our global multicenter experience with CRS-associated coagulopathy after tisagenlecleucel therapy in 137 patients with relapsed or refractory B-cell acute lymphoblastic leukemia from the ELIANA and ENSIGN trials. These trials included clinical guidelines for fibrinogen replacement during CRS-associated coagulopathy. Hypofibrinogenemia requiring replacement was observed only in patients with severe CRS. A higher percentage of patients who required replacement were <10 years old, compared with those who did not require replacement. Twenty-three patients received replacement for hypofibrinogenemia (<1.5 g/L); 9 of them developed marked hypofibrinogenemia (<1 g/L). Very low fibrinogen levels (<1 g/L) were documented in patients before maximal CRS (n = 1), during maximal CRS (n = 7), and at CRS improvement (n = 1). Although hypofibrinogenemia was the most clinically significant coagulopathy, some patients also developed prolonged prothrombin time and activated partial thromboplastin time and increased international normalized ratio, further increasing the risk of bleeding. Hypofibrinogenemia was effectively managed using fibrinogen concentrate or cryoprecipitate replacement; severe (grade 4) bleeding events were rare (n = 2). CRS-associated coagulopathy with hypofibrinogenemia is manageable according to empiric guidelines of fibrinogen replacement for CAR-T trials. Fibrinogen concentrate should be used when cryoprecipitate is not reliably available. Monitoring fibrinogen levels in patients with moderate or severe CRS is essential for avoiding potentially fatal bleeding events. These trials were registered at www.clinicaltrials.gov as #NCT02435849 and #NCT02228096.
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http://dx.doi.org/10.1182/bloodadvances.2020002757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839371PMC
January 2021

Beyond the storm - subacute toxicities and late effects in children receiving CAR T cells.

Nat Rev Clin Oncol 2021 06 25;18(6):363-378. Epub 2021 Jan 25.

Pediatric Oncology Branch, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA.

As clinical advances with chimeric antigen receptor (CAR) T cells are increasingly described and the potential for extending their therapeutic benefit grows, optimizing the implementation of this therapeutic modality is imperative. The recognition and management of cytokine release syndrome (CRS) marked a milestone in this field; however, beyond the understanding gained in treating CRS, a host of additional toxicities and/or potential late effects of CAR T cell therapy warrant further investigation. A multicentre initiative involving experts in paediatric cell therapy, supportive care and/or study of late effects from cancer and haematopoietic stem cell transplantation was convened to facilitate the comprehensive study of extended CAR T cell-mediated toxicities and establish a framework for new systematic investigations of CAR T cell-related adverse events. Together, this group identified six key focus areas: extended monitoring of neurotoxicity and neurocognitive function, psychosocial considerations, infection and immune reconstitution, other end organ toxicities, evaluation of subsequent neoplasms, and strategies to optimize remission durability. Herein, we present the current understanding, gaps in knowledge and future directions of research addressing these CAR T cell-related outcomes. This systematic framework to study extended toxicities and optimization strategies will facilitate the translation of acquired experience and knowledge for optimal application of CAR T cell therapies.
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http://dx.doi.org/10.1038/s41571-020-00456-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8335746PMC
June 2021

Risk-Adapted Preemptive Tocilizumab to Prevent Severe Cytokine Release Syndrome After CTL019 for Pediatric B-Cell Acute Lymphoblastic Leukemia: A Prospective Clinical Trial.

J Clin Oncol 2021 03 8;39(8):920-930. Epub 2021 Jan 8.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

Purpose: To 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.

Methods: Children 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.

Results: The 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 ( < .001), but persistence was not different ( = .73). Event-free and overall survival were worse in the HTBC ( = .004, < .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 ( = .18).

Conclusion: Risk-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.
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http://dx.doi.org/10.1200/JCO.20.02477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8462622PMC
March 2021

Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immune effector cell-related adverse events.

J Immunother Cancer 2020 12;8(2)

Cancer Immunotherapy Program, Division of Oncology, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, Pennsylvania, USA

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.
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http://dx.doi.org/10.1136/jitc-2020-001511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745688PMC
December 2020

Diagnostic biomarkers to differentiate sepsis from cytokine release syndrome in critically ill children.

Blood Adv 2020 10;4(20):5174-5183

Division of Hematology and Oncology, Children's Hospital of Philadelphia, Philadelphia, PA.

Chimeric antigen receptor (CAR) T-cells directed against CD19 have drastically altered outcomes for children with relapsed and refractory acute lymphoblastic leukemia (r/r ALL). Pediatric patients with r/r ALL treated with CAR-T are at increased risk of both cytokine release syndrome (CRS) and sepsis. We sought to investigate the biologic differences between CRS and sepsis and to develop predictive models which could accurately differentiate CRS from sepsis at the time of critical illness. We identified 23 different cytokines that were significantly different between patients with sepsis and CRS. Using elastic net prediction modeling and tree classification, we identified cytokines that were able to classify subjects as having CRS or sepsis accurately. A markedly elevated interferon γ (IFNγ) or a mildly elevated IFNγ in combination with a low IL1β were associated with CRS. A normal to mildly elevated IFNγ in combination with an elevated IL1β was associated with sepsis. This combination of IFNγ and IL1β was able to categorize subjects as having CRS or sepsis with 97% accuracy. As CAR-T therapies become more common, these data provide important novel information to better manage potential associated toxicities.
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http://dx.doi.org/10.1182/bloodadvances.2020002592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594400PMC
October 2020

Lingering effects of chemotherapy on mature T cells impair proliferation.

Blood Adv 2020 10;4(19):4653-4664

Division of Oncology, Department of Pediatrics.

Engineered T-cell therapies have demonstrated impressive clinical responses in patients with hematologic malignancies. Despite this efficacy, many patients have a transient persistence of T cells, which can be correlated with transient clinical response. Translational data on T cells from pediatric cancer patients shows a progressive decline in chimeric antigen receptor (CAR) suitability with cumulative chemotherapy regardless of regimen. We investigated the effects of chemotherapy on surviving T cells in vitro, describing residual deficits unique to each agent including mitochondrial damage and metabolic alterations. In the case of cyclophosphamide but not doxorubicin or cytarabine, these effects could be reversed with N-acetylcysteine. Specifically, we observed that surviving T cells could be stimulated, expanded, and transduced with CARs with preserved short-term cytolytic function but at far lower numbers and with residual metabolic deficits. These data have implications for understanding the effects of chemotherapy on mature T cells later collected for adoptive cell therapy, as chemotherapy-exposed T cells may have lingering dysfunction that affects ex vivo adoptive cell therapy manufacturing techniques and, ultimately, clinical efficacy.
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http://dx.doi.org/10.1182/bloodadvances.2020001797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556159PMC
October 2020

Analysis of Time to Complete Response after Defibrotide Initiation in Patients with Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome after Hematopoietic Cell Transplantation.

Transplant Cell Ther 2021 01 17;27(1):88.e1-88.e6. Epub 2020 Sep 17.

Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) is a potentially life-threatening complication that occurs after hematopoietic cell transplantation (HCT). The mortality associated with untreated VOD/SOS with multiorgan dysfunction (MOD) has been reported to be >80%. The recommended dose of defibrotide is 6.25 mg/kg every 6 hours, administered as a 2-hour i.v. infusion, for a minimum of 21 days or until resolution of VOD/SOS signs and symptoms. The objective of this analysis was to evaluate the time to complete response (CR) in patients with post-HCT VOD/SOS treated with defibrotide. The time to defibrotide discontinuation due to a CR served as a surrogate for time to CR in an expanded access study (T-IND; ClinicalTrials.gov NCT00628498; n = 1000), and was analyzed separately from the time to CR data pooled from a phase 2 randomized dose-finding study (NCT00003966; n = 74 patients who received 25 mg/kg/day) and a phase 3 historically controlled study (NCT00358501; n = 102). For all studies, a CR was defined as total serum bilirubin <2 mg/dL with resolution of VOD/SOS-related MOD (renal and/or pulmonary dysfunction); the phase 2 study also required resolution of central nervous system dysfunction. In the T-IND, 390 patients discontinued treatment due to a CR and had sufficient data for analysis. The median time to discontinuation was 22 days (range, 2 to 64 days). Discontinuation due to CR occurred beyond 21 days in 235 patients (60%) and beyond 28 days in 57 patients (15%). The pooled phase 2 and 3 studies included 60 patients who achieved a CR, with a median time to CR of 24.5 days (range, 7 to 123 days). A CR was achieved beyond 21 days in 32 patients (53%) and beyond 28 days in 24 patients (40%). The Kaplan-Meier estimate of day +100 survival rate was substantially higher in patients who discontinued due to a CR compared with those who did not (92.5% versus 37.3%). Treatment-emergent adverse events occurred in 185 of 390 patients (47%) who discontinued due to a CR in the T-IND and in 55 of 60 patients (92%) who achieved a CR in the pooled phase 2 and 3 studies, and rates did not differ according to duration of treatment (≤21 days versus >21 days). Taken together, these results highlight the importance of continued defibrotide therapy until resolution of VOD/SOS signs and symptoms, as currently indicated in the approved product labels, which may occur beyond the recommended minimum of 21 days.
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http://dx.doi.org/10.1016/j.bbmt.2020.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8549529PMC
January 2021

Multisystem inflammatory syndrome in children and COVID-19 are distinct presentations of SARS-CoV-2.

J Clin Invest 2020 11;130(11):5967-5975

Immune Dysregulation Frontier Program.

BACKGROUNDInitial reports from the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic described children as being less susceptible to coronavirus disease 2019 (COVID-19) than adults. Subsequently, a severe and novel pediatric disorder termed multisystem inflammatory syndrome in children (MIS-C) emerged. We report on unique hematologic and immunologic parameters that distinguish between COVID-19 and MIS-C and provide insight into pathophysiology.METHODSWe prospectively enrolled hospitalized patients with evidence of SARS-CoV-2 infection and classified them as having MIS-C or COVID-19. Patients with COVID-19 were classified as having either minimal or severe disease. Cytokine profiles, viral cycle thresholds (Cts), blood smears, and soluble C5b-9 values were analyzed with clinical data.RESULTSTwenty patients were enrolled (9 severe COVID-19, 5 minimal COVID-19, and 6 MIS-C). Five cytokines (IFN-γ, IL-10, IL-6, IL-8, and TNF-α) contributed to the analysis. TNF-α and IL-10 discriminated between patients with MIS-C and severe COVID-19. The presence of burr cells on blood smears, as well as Cts, differentiated between patients with severe COVID-19 and those with MIS-C.CONCLUSIONPediatric patients with SARS-CoV-2 are at risk for critical illness with severe COVID-19 and MIS-C. Cytokine profiling and examination of peripheral blood smears may distinguish between patients with MIS-C and those with severe COVID-19.FUNDINGFinancial support for this project was provided by CHOP Frontiers Program Immune Dysregulation Team; National Institute of Allergy and Infectious Diseases; National Cancer Institute; the Leukemia and Lymphoma Society; Cookies for Kids Cancer; Alex's Lemonade Stand Foundation for Childhood Cancer; Children's Oncology Group; Stand UP 2 Cancer; Team Connor; the Kate Amato Foundations; Burroughs Wellcome Fund CAMS; the Clinical Immunology Society; the American Academy of Allergy, Asthma, and Immunology; and the Institute for Translational Medicine and Therapeutics.
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http://dx.doi.org/10.1172/JCI140970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598044PMC
November 2020

Thoracic duct lymphatic fluid harbors phenotypically naive T cells for use in adoptive T-cell therapy.

Cytotherapy 2020 10 1;22(10):529-535. Epub 2020 Jul 1.

Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.

Background Aims: Manufacturing of potent chimeric antigen receptor (CAR) T cells requires phenotypically naive and early memory T cells. We hypothesized lymphatic fluid collected from the thoracic duct of children would serve as a unique reservoir for early T cells, which could then be used for CAR T-cell therapy.

Methods: We evaluated lymphatic fluid collected from 25 pediatric patients undergoing thoracic duct cannulation for other clinical indications.

Results: Lymphatic fluid in the thoracic duct was rich in T cells, with higher percentage of naive and stem central memory T-cell subsets compared with paired blood samples. T cells from lymphatic fluid showed decreased negative checkpoint regulators on the surface and increased rapid expansion with bead activation. Creation of CD19-directed CAR T cells from blood and lymphatic T cells showed similar lentiviral transduction properties, but CAR T cells generated from lymphatic fluid produced superior cytotoxicity in a murine leukemia model because they were able to achieve equivalent tumor eradication at lower doses.

Conclusions: These results are the first characterization of T cells from the thoracic duct of pediatric patients and suggest an alternative approach for manufacturing of cellular therapy that will improve both expansion and cytotoxic effect.
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http://dx.doi.org/10.1016/j.jcyt.2020.05.004DOI Listing
October 2020
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