FOXO1 is a master regulator of memory programming in CAR T cells.

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2-6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.

Exagamglogene Autotemcel for Transfusion-Dependent ß-Thalassemia.

BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis through ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of the erythroid-specific enhancer region of BCL11A in autologous CD34+ hematopoietic stem and progenitor cells (HSPCs). METHODS: We conducted an open-label, single-group, phase 3 study of exa-cel in patients 12 to 35 years of age with transfusion-dependent ß-thalassemia and a ß0/ß0, ß0/ß0-like, or non-ß0/ß0-like genotype. CD34+ HSPCs were edited by means of CRISPR-Cas9 with a guide mRNA. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was transfusion independence, defined as a weighted average hemoglobin level of 9 g per deciliter or higher without red-cell transfusion for at least 12 consecutive months. Total and fetal hemoglobin concentrations and safety were also assessed. RESULTS: A total of 52 patients with transfusion-dependent ß-thalassemia received exa-cel and were included in this prespecified interim analysis; the median follow-up was 20.4 months (range, 2.1 to 48.1). Neutrophils and platelets engrafted in each patient. Among the 35 patients with sufficient follow-up data for evaluation, transfusion independence occurred in 32 (91%; 95% confidence interval, 77 to 98; P<0.001 against the null hypothesis of a 50% response). During transfusion independence, the mean total hemoglobin level was 13.1 g per deciliter and the mean fetal hemoglobin level was 11.9 g per deciliter, and fetal hemoglobin had a pancellular distribution (≥94% of red cells). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No deaths or cancers occurred. CONCLUSIONS: Treatment with exa-cel, preceded by myeloablation, resulted in transfusion independence in 91% of patients with transfusion-dependent ß-thalassemia. (Supported by Vertex Pharmaceuticals and CRISPR Therapeutics; CLIMB THAL-111 ClinicalTrials.gov number, NCT03655678.).

Exagamglogene Autotemcel for Severe Sickle Cell Disease.

BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A. METHODS: We conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed. RESULTS: A total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred. CONCLUSIONS: Treatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.).

Quadriparesis and paraparesis following chimeric antigen receptor T-cell (CART) therapy in children and adolescents.

Immune effector cell-associated neurotoxicity syndrome (ICANS) is a common but potentially severe adverse event associated with chimeric antigen receptor T-cell (CART) therapy characterized by the development of acute neurologic symptoms following CART infusion. ICANS encompasses a wide clinical spectrum typified by mild to severe encephalopathy, seizures and/or cerebral edema. As more patients have been treated with CART new ICANS phenomenology has emerged. We present the clinical course of five children who developed acute onset of quadriparesis or paraparesis associated with abnormal brain and/or spine neuroimaging after infusion of CD19 or CD22-directed CART, adverse events not previously reported in children. Orthogonal data from autopsy studies, cerebrospinal fluid (CSF) flow cytometry and CSF proteomics/cytokine profiling demonstrated chronic white matter destruction, but a notable lack of inflammatory pathologic changes and cell populations. Instead, children with quadriparesis or paraparesis post-CART therapy had lower levels of pro-inflammatory cytokines such as interferon gamma (IFN), CCL17, CCL23, and CXCL10 than those who did not develop quadriparesis or paraparesis. Taken together, these findings imply a non-inflammatory source of this newly described ICANS phenomenon in children. The pathophysiology of some neurologic symptoms following CART may therefore have a more complex etiology than exclusive T-cell activation and excessive cytokine production.

Impact of poverty and neighborhood opportunity on outcomes for children treated with CD19-directed CAR T-cell therapy.

Children living in poverty experience excessive relapse and death from newly diagnosed acute lymphoblastic leukemia (ALL). The influence of household poverty and neighborhood social determinants on outcomes from chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory (r/r) leukemia is poorly described. We identified patients with r/r CD19+ ALL/lymphoblastic lymphoma treated on CD19-directed CAR T-cell clinical trials or with commercial tisagenlecleucel from 2012 to 2020. Socioeconomic status (SES) was proxied at the household level, with poverty exposure defined as Medicaid-only insurance. Low-neighborhood opportunity was defined by the Childhood Opportunity Index. Among 206 patients aged 1 to 29, 35.9% were exposed to household poverty, and 24.9% had low-neighborhood opportunity. Patients unexposed to household poverty or low-opportunity neighborhoods were more likely to receive CAR T-cell therapy with a high disease burden (>25%), a disease characteristic associated with inferior outcomes, as compared with less advantaged patients (38% vs 30%; 37% vs 26%). Complete remission (CR) rate was 93%, with no significant differences by household poverty (P = .334) or neighborhood opportunity (P = .504). In multivariate analysis, patients from low-opportunity neighborhoods experienced an increased hazard of relapse as compared with others (P = .006; adjusted hazard ratio [HR], 2.3; 95% confidence interval [CI], 1.3-4.1). There was no difference in hazard of death (P = .545; adjusted HR, 1.2; 95% CI, 0.6-2.4). Among children who successfully receive CAR T-cell therapy, CR and overall survival are equitable regardless of proxied SES and neighborhood opportunity. Children from more advantaged households and neighborhoods receive CAR T-cell therapy with a higher disease burden. Investigation of multicenter outcomes and access disparities outside of clinical trial settings is warranted.

CRISPR-Cas9 Gene Editing for Sickle Cell Disease and ß-Thalassemia.

Transfusion-dependent ß-thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells. We performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the BCL11A erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients - one with TDT and the other with SCD - received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same BCL11A enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex Pharmaceuticals; ClinicalTrials.gov numbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.).

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

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.

Cytosine base editing enables quadruple-edited allogeneic CART cells for T-ALL.

Allogeneic chimeric antigen receptor T-cell (CART) therapies require multiple gene edits to be clinically tractable. Most allogeneic CARTs 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% and 99% efficiency to silence gene expression without creating DSBs, greatly reducing or eliminating undesired editing outcomes following multiplexed editing as compared with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). Using CBE, we developed 7CAR8, a CD7-directed allogeneic CART created using 4 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.

Bone marrow fibrosis is associated with non-response to CD19 CAR T-cell therapy in B-acute lymphoblastic leukemia.

CD19 directed CAR T-cell therapy is used to treat relapsed/refractory B-cell acute lymphoblastic leukemia. The role of the pre-CAR bone marrow (BM) stromal microenvironment in determining response to CAR T-cell therapy has been understudied. We performed whole transcriptome analysis, reticulin fibrosis assessment and CD3 T-cell infiltration on BM core biopsies from pre- and post-CAR timepoints for 61 patients, as well as on a cohort of 54 primary B-ALL samples. Pathways of fibrosis, extracellular matrix development, and associated transcription factors AP1 and TGF-ß3, were enriched and upregulated in nonresponders (NR) even prior to CAR T cell therapy. NR showed significantly higher levels of BM fibrosis compared to complete responders by both clinical reticulin assessment and AI-assisted digital image scoring. CD3+ T cells showed a trend toward lower infiltration in NR. NR had significantly higher levels of pre-CAR fibrosis compared to primary B-ALL. High levels of fibrosis were associated with lower overall survival after CAR T-cell therapy. In conclusion, BM fibrosis is a novel mechanism mediating nonresponse to CD19-directed CAR T-cell therapy in B-ALL. A widely used clinically assay for quantitating myelofibrosis can be repurposed to determine patients at high risk of non-response. Genes and pathways associated with BM fibrosis are a potential target to improve response.

Impact of diagnostic and end-of-induction Curie scores with tandem high-dose chemotherapy and autologous transplants for metastatic high-risk neuroblastoma: A report from the Children's Oncology Group.

BACKGROUND: Diagnostic mIBG (meta-iodobenzylguanidine) scans are an integral component of response assessment in children with high-risk neuroblastoma. The role of end-of-induction (EOI) Curie scores (CS) was previously described in patients undergoing a single course of high-dose chemotherapy (HDC) and autologous hematopoietic cell transplant (AHCT) as consolidation therapy. OBJECTIVE: We now examine the prognostic significance of CS in patients randomized to tandem HDC and AHCT on the Children's Oncology Group (COG) trial ANBL0532. STUDY DESIGN: A retrospective analysis of mIBG scans obtained from patients enrolled in COG ANBL0532 was performed. Evaluable patients had mIBG-avid, International Neuroblastoma Staging System (INSS) stage 4 disease, did not progress during induction therapy, consented to consolidation randomization, and received either single or tandem HDC (n = 80). Optimal CS cut points maximized the outcome difference (≤CS vs. >CS cut-off) according to the Youden index. RESULTS: For recipients of tandem HDC, the optimal cut point at diagnosis was CS = 12, with superior event-free survival (EFS) from study enrollment for patients with CS ≤ 12 (3-year EFS 74.2% ± 7.9%) versus CS > 12 (59.2% ± 7.1%) (p = .002). At EOI, the optimal cut point was CS = 0, with superior EOI EFS for patients with CS = 0 (72.9% ± 6.4%) versus CS > 0 (46.5% ± 9.1%) (p = .002). CONCLUSION: In the setting of tandem transplantation for children with high-risk neuroblastoma, CS at diagnosis and EOI may identify a more favorable patient group. Patients treated with tandem HDC who exhibited a CS ≤ 12 at diagnosis or CS = 0 at EOI had superior EFS compared to those with CS above these cut points.

Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia.

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 .).

A safety and feasibility trial of 131 I-MIBG in newly diagnosed high-risk neuroblastoma: A Children's Oncology Group study.

INTRODUCTION: 131 I-meta-iodobenzylguanidine (131 I-MIBG) is effective in relapsed neuroblastoma. The Children's Oncology Group (COG) conducted a pilot study (NCT01175356) to assess tolerability and feasibility of induction chemotherapy followed by 131 I- MIBG therapy and myeloablative busulfan/melphalan (Bu/Mel) in patients with newly diagnosed high-risk neuroblastoma. METHODS: Patients with MIBG-avid high-risk neuroblastoma were eligible. After the first two patients to receive protocol therapy developed severe sinusoidal obstruction syndrome (SOS), the trial was re-designed to include an 131 I-MIBG dose escalation (12, 15, and 18 mCi/kg), with a required 10-week gap before Bu/Mel administration. Patients who completed induction chemotherapy were evaluable for assessment of 131 I-MIBG feasibility; those who completed 131 I-MIBG therapy were evaluable for assessment of 131 I-MIBG + Bu/Mel feasibility. RESULTS: Fifty-nine of 68 patients (86.8%) who completed induction chemotherapy received 131 I-MIBG. Thirty-seven of 45 patients (82.2%) evaluable for 131 I-MIBG + Bu/Mel received this combination. Among those who received 131 I-MIBG after revision of the study design, one patient per dose level developed severe SOS. Rates of moderate to severe SOS at 12, 15, and 18 mCi/kg were 33.3%, 23.5%, and 25.0%, respectively. There was one toxic death. The 131 I-MIBG and 131 I-MIBG+Bu/Mel feasibility rates at the 15 mCi/kg dose level designated for further study were 96.7% (95% CI: 83.3%-99.4%) and 81.0% (95% CI: 60.0%-92.3%). CONCLUSION: This pilot trial demonstrated feasibility and tolerability of administering 131 I-MIBG followed by myeloablative therapy with Bu/Mel to newly diagnosed children with high-risk neuroblastoma in a cooperative group setting, laying the groundwork for a cooperative randomized trial (NCT03126916) testing the addition of 131 I-MIBG during induction therapy.

Chimeric Antigen Receptor T Cell Therapy During the COVID-19 Pandemic.

The SARS-CoV-2 coronavirus (COVID-19) pandemic has significantly impacted the delivery of cellular therapeutics, including chimeric antigen receptor (CAR) T cells. This impact has extended beyond patient care to include logistics, administration, and distribution of increasingly limited health care resources. Based on the collective experience of the CAR T-cell Consortium investigators, we review and address several questions and concerns regarding cellular therapy administration in the setting of COVID-19 and make general recommendations to address these issues. Specifically, we address (1) necessary resources for safe administration of cell therapies; (2) determinants of cell therapy utilization; (3) selection among patients with B cell non-Hodgkin lymphomas and B cell acute lymphoblastic leukemia; (4) supportive measures during cell therapy administration; (5) use and prioritization of tocilizumab; and (6) collaborative care with referring physicians. These recommendations were carefully formulated with the understanding that resource allocation is of the utmost importance, and that the decision to proceed with CAR T cell therapy will require extensive discussion of potential risks and benefits. Although these recommendations are fluid, at this time it is our opinion that the COVID-19 pandemic should not serve as reason to defer CAR T cell therapy for patients truly in need of a potentially curative therapy.

Partially CD3+-Depleted Unrelated and Haploidentical Donor Peripheral Stem Cell Transplantation Has Favorable Graft-versus-Host Disease and Survival Rates in Pediatric Hematologic Malignancy.

Most children who may benefit from stem cell transplantation lack a matched related donor. Alternative donor transplantations with an unrelated donor (URD) or a partially matched related donor (PMRD) carry an increased risk of graft-versus-host-disease (GVHD) and mortality compared with matched related donor transplantations. We hypothesized that a strategy of partial CD3+/CD19+ depletion for URD or PMRD peripheral stem cell transplantation (PSCT) would attenuate the risks of GVHD and mortality. We enrolled 84 pediatric patients with hematologic malignancies at the Children's Hospital of Philadelphia and the Children's Hospital of Wisconsin between April 2005 and February 2015. Two patients (2.4%) experienced primary graft failure. Relapse occurred in 23 patients (27.4%; cumulative incidence 26.3%), and 17 patients (20.2%) experienced nonrelapse mortality (NRM). Grade III-IV acute GVHD was observed in 18 patients (21.4%), and chronic GVHD was observed and graded as limited in 24 patients (35.3%) and extensive in 8 (11.7%). Three-year overall survival (OS) was 61.8% (95% confidence interval [CI], 50.2% to 71.4%) and event-free survival (EFS) was 52.0% (95% CI, 40.3% to 62.4%). Age ≥15 years was associated with decreased OS (P= .05) and EFS (P= .05). Relapse was more common in children in second complete remission (P = .03). Partially CD3+-depleted alternative donor PSCT NRM, OS, and EFS compare favorably with previously published studies of T cell-replete PSCT. Historically, T cell-replete PSCT has been associated with a higher incidence of extensive chronic GVHD compared with limited chronic GVHD, which may explain the comparatively low relapse and NRM rates in our study cohort despite similar overall rates of chronic GVHD. Partial T cell depletion may expand donor options for children with malignant transplantation indications lacking a matched related donor by mitigating, but not eliminating, chronic GVHD.

Pooled analysis of Day 100 survival for defibrotide-treated patients with hepatic veno-occlusive disease/sinusoidal obstruction syndrome and ventilator or dialysis dependence following haematopoietic cell transplantation.

For patients with untreated hepatic veno-occlusive disease (VOD)/sinusoidal obstruction syndrome (SOS) with multi-organ dysfunction (MOD), mortality is >80%. We conducted a pooled analysis of three studies that assessed Day 100 survival in relationship to MOD severity, with dialysis and/or ventilator dependence representing the most severe organ dysfunction. All patients in the analysis were diagnosed using Baltimore criteria/biopsy. This analysis of patients with VOD/SOS and MOD after haematopoietic cell transplantation (HCT; n = 651) demonstrated higher Day 100 survival rates amongst defibrotide-treated patients with VOD/SOS with less versus more severe forms of MOD. Even patients with severe forms of MOD post-HCT benefitted from defibrotide.

Nonviral RNA chimeric antigen receptor-modified T cells in patients with Hodgkin lymphoma.

Chimeric antigen receptor (CAR)-modified T cells are being investigated in many settings, including classical Hodgkin lymphoma (cHL). The unique biology of cHL, characterized by scant Hodgkin and Reed-Sternberg (HRS) cells within an immunosuppressive tumor microenvironment (TME), may pose challenges for cellular therapies directly targeting antigens expressed on HRS cells. We hypothesized that eradicating CD19+ B cells within the TME and the putative circulating CD19+ HRS clonotypic cells using anti-CD19-directed CAR-modified T cells (CART19) may indirectly affect HRS cells, which do not express CD19. Here we describe our pilot trial using CART19 in patients with relapsed or refractory cHL. To limit potential toxicities, we used nonviral RNA CART19 cells, which are expected to express CAR protein for only a few days, as opposed to CART19 generated by viral vector transduction, which expand in vivo and retain CAR expression. All 5 enrolled patients underwent successful manufacturing of nonviral RNA CART19, and 4 were infused with protocol-specified cell dose. There were no severe toxicities. Responses were seen, but these were transient. To our knowledge, this is the first CART19 clinical trial to use nonviral RNA gene delivery. This trial was registered at www.clinicaltrials.gov as #NCT02277522 (adult) and #NCT02624258 (pediatric).

Preclinical efficacy of daratumumab in T-cell acute lymphoblastic leukemia.

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

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

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.

Subcutaneous immunoglobulin replacement following CD19-specific chimeric antigen receptor T-cell therapy for B-cell acute lymphoblastic leukemia in pediatric patients.

Twenty-eight patients were maintained on subcutaneous immunoglobulin replacement for persistent B-cell aplasia and agammaglobulinemia following CD19-targeted chimeric antigen receptor T-cell therapy for B-cell lymphoblastic leukemia. Patients were transitioned from intravenous to subcutaneous immunoglobulin replacement at a median of 11.5 months (range, 4-20). Increasing serum IgG level was significantly associated with a lower rate of sinopulmonary infection (P = 0.0072). The median serum IgG level during infection-free periods was 1000 mg/dL (range, 720-1430), which was significantly higher than IgG levels in patients with sinopulmonary infections. As such, we recommend maintaining a goal IgG level > 1000 mg/dL to provide optimal protection.