INSPIRED Symposium Part 5: Expanding the Use of CAR T Cells in Children and Young Adults.

Chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable efficacy in relapsed/refractory (r/r) B cell malignancies, including in pediatric patients with acute lymphoblastic leukemia (ALL). Expanding this success to other hematologic and solid malignancies is an area of active research and, although challenges remain, novel solutions have led to significant progress over the past decade. Ongoing clinical trials for CAR T cell therapy for T cell malignancies and acute myeloid leukemia (AML) have highlighted challenges, including antigen specificity with off-tumor toxicity and persistence concerns. In T cell malignancies, notable challenges include CAR T cell fratricide and prolonged T cell aplasia, which are being addressed with strategies such as gene editing and suicide switch technologies. In AML, antigen identification remains a significant barrier, due to shared antigens across healthy hematopoietic progenitor cells and myeloid blasts. Strategies to limit persistence and circumvent the immunosuppressive tumor microenvironment (TME) created by AML are also being explored. CAR T cell therapies for central nervous system and solid tumors have several challenges, including tumor antigen heterogeneity, immunosuppressive and hypoxic TME, and potential for off-target toxicity. Numerous CAR T cell products have been designed to overcome these challenges, including "armored" CARs and CAR/T cell receptor (TCR) hybrids. Strategies to enhance CAR T cell delivery, augment CAR T cell performance in the TME, and ensure the safety of these products have shown promising results. In this manuscript, we will review the available evidence for CAR T cell use in T cell malignancies, AML, central nervous system (CNS), and non-CNS solid tumor malignancies, and recommend areas for future research.

Acute kidney injury following treatment with CD19-specific CAR T-cell therapy in children, adolescent, and young adult patients with B-cell acute lymphoblastic leukemia.

BACKGROUND: CD19-specific chimeric antigen receptor (CAR) T-cell therapy has shown promising disease responses in patients with high-risk B-cell malignancies. However, its use may be related to complications such as immune-mediated complications, infections, and end-organ dysfunction. The incidence of post-CAR T-cell therapy acute kidney injury (AKI) in the children, adolescent, and young adult (CAYA) patient population is largely unreported. METHODS: The objectives of this study were to determine the incidence of AKI in CAYA patients with high-risk B-cell malignancies treated with CD19-CAR T-cell therapy, evaluate potential risk factors for developing AKI, and determine patterns of kidney function recovery. We conducted a retrospective analysis of 34 CAYA patients treated with CD19-CAR T-cell at a single institution. RESULTS: There was a cumulative incidence of any grade AKI by day 30 post-infusion of 20% (n = 7), with four cases being severe AKI (stages 2-3) and one patient requiring kidney replacement therapy. All episodes of AKI developed within the first 14 days after receiving CAR T-cell therapy and 50% of patients with AKI recovered kidney function to baseline within 30 days post-infusion. No evaluated pre-treatment risk factors were associated with the development of subsequent AKI; there was an association between AKI and cytokine release syndrome and neurotoxicity. We conclude that the risk of developing AKI following CD19-CAR T-cell therapy is highest early post-infusion, with most cases of AKI being severe. CONCLUSIONS: Frequent monitoring to facilitate early recognition and subsequent management of kidney complications after CD19-CAR T-cell therapy may reduce the severity of AKI in the CAYA patient population.

INSPIRED Symposium Part 3: Prevention and Management of Pediatric Chimeric Antigen Receptor T Cell-Associated Emergent Toxicities.

Chimeric antigen receptor (CAR) T cell (CAR-T) therapy has emerged as a revolutionary cancer treatment modality, particularly in children and young adults with B cell malignancies. Through clinical trials and real-world experience, much has been learned about the unique toxicity profile of CAR-T therapy. The past decade brought advances in identifying risk factors for severe inflammatory toxicities, investigating preventive measures to mitigate these toxicities, and exploring novel strategies to manage refractory and newly described toxicities, infectious risks, and delayed effects, such as cytopenias. Although much progress has been made, areas needing further improvements remain. Limited guidance exists regarding initial administration of tocilizumab with or without steroids and the management of inflammatory toxicities refractory to these treatments. There has not been widespread adoption of preventive strategies to mitigate inflammation in patients at high risk of severe toxicities, particularly children. Additionally, the majority of research related to CAR-T toxicity prevention and management has focused on adult populations, with only a few pediatric-specific studies published to date. Given that children and young adults undergoing CAR-T therapy represent a unique population with different underlying disease processes, physiology, and tolerance of toxicities than adults, it is important that studies be conducted to evaluate acute, delayed, and long-term toxicities following CAR-T therapy in this younger age group. In this pediatric-focused review, we summarize key findings on CAR-T therapy-related toxicities over the past decade, highlight emergent CAR-T toxicities, and identify areas of greatest need for ongoing research.

Preventing relapse after CD19 CAR T-cell therapy for pediatric ALL: the role of transplant and enhanced CAR T cells.

CD19-specific chimeric antigen receptor (CAR) T-cell therapy has become an integral part of our treatment armamentarium for pediatric patients with relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, despite initial remission rates of greater than 80%, durable remission occurs in only 40% to 50% of patients. In this review we summarize our current knowledge of the role of consolidative hematopoietic cell transplantation in the management of pediatric patients who achieved a minimal residual disease-negative complete response post CD19 CAR T-cell therapy. In addition, we review approaches to enhance effector function CD19 CAR T cells, focusing on how to improve persistence and prevent the emergence of CD19- B-ALL blasts.

Latent human herpesvirus 6 is reactivated in CAR T cells.

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.

Acute kidney injury following treatment with CD19-specific CAR T-cell therapy in children, adolescent and young adult patients with B-cell acute lymphoblastic leukemia.

CD19-specific chimeric antigen receptor (CAR) T-cell therapy has shown promising disease responses in patients with high-risk B-cell malignancies. Treatment with CD19-CAR T-cell therapy is also associated with the risk of morbidity and mortality, primarily related to immune-mediated complications (cytokine release syndrome [CRS] and neurotoxicity [NTX]), infections, and end-organ dysfunction. Despite these well-described systemic toxicities, the incidence of post-CAR T-cell therapy acute kidney injury (AKI) in the children, adolescent and young adult (CAYA) patient population is largely unreported. The objectives of this study were to determine the incidence of AKI in CAYA patients with high-risk B-cell malignancies treated with CD19-CAR T-cell therapy, evaluate potential risk factors for developing AKI, and determine patterns of kidney function recovery. In this retrospective analysis of 34 CAYA patients treated with CD19-CAR T-cell at a single institution, we found a cumulative incidence of any grade AKI by day 30 post-infusion of 20% (n=7), with 4 cases being severe AKI (Stage 2-3) and one patient requiring kidney replacement therapy. All episodes of AKI developed within the first 14 days after receiving CAR T-cell therapy and 50% of patients with AKI recovered kidney function to baseline within 30 days post-infusion. No evaluated pre-treatment risk factors were associated with the development of subsequent AKI; there was an association between AKI and CRS and NTX. We conclude that the risk of developing AKI following CD19-CAR T-cell therapy is highest early post-infusion, with most cases of AKI being severe. Although most patients with AKI in our cohort had recovery of kidney function, frequent monitoring to facilitate early recognition and subsequent management of kidney complications after CD19-CAR T-cell therapy may reduce the severity of AKI in the CAYA patient population.

Integrome signatures of lentiviral gene therapy for SCID-X1 patients.

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.

Integration of Palliative Care in Hematopoietic Cell Transplant: Pediatric Patient and Parent Needs and Attitudes.

CONTEXT: Early integration of palliative care (PC) in hematopoietic cell transplantation (HCT) has demonstrated benefits, yet barriers remain, including perceived lack of patient/caregiver receptivity despite no data on attitudes toward PC and limited patient/caregiver reported outcomes in pediatric HCT. OBJECTIVES: This study aimed to evaluate perceived symptom burden and patient/parent attitudes toward early PC integration in pediatric HCT. METHODS: Following IRB approval, consent/assent, eligible participants were surveyed at St. Jude Children's Research Hospital including English-speaking patients aged 10-17, 1-month to 1-year from HCT, and their parents/primary-caregivers, as well as parent/primary-caregivers of living HCT recipients

Immune Effector Cell-Associated Hemophagocytic Lymphohistiocytosis-Like Syndrome.

T cell-mediated hyperinflammatory responses, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), are now well-established toxicities of chimeric antigen receptor (CAR) T cell therapy. As the field of CAR T cells advances, however, there is increasing recognition that hemophagocytic lymphohistiocytosis (HLH)-like toxicities following CAR T cell infusion are occurring broadly across patient populations and CAR T cell constructs. Importantly, these HLH-like toxicities are often not as directly associated with CRS and/or its severity as initially described. This emergent toxicity, however ill-defined, is associated with life-threatening complications, creating an urgent need for improved identification and optimal management. With the goal of improving patient outcomes and formulating a framework to characterize and study this HLH-like syndrome, we established an American Society for Transplantation and Cellular Therapy panel composed of experts in primary and secondary HLH, pediatric and adult HLH, infectious disease, rheumatology and hematology, oncology, and cellular therapy. Through this effort, we provide an overview of the underlying biology of classical primary and secondary HLH, explore its relationship with similar manifestations following CAR T cell infusions, and propose the term "immune effector cell-associated HLH-like syndrome (IEC-HS)" to describe this emergent toxicity. We also delineate a framework for identifying IEC-HS and put forward a grading schema that can be used to assess severity and facilitate cross-trial comparisons. Additionally, given the critical need to optimize outcomes for patients experiencing IEC-HS, we provide insight into potential treatment approaches and strategies to optimize supportive care and delineate alternate etiologies that should be considered in a patient presenting with IEC-HS. By collectively defining IEC-HS as a hyperinflammatory toxicity, we can now embark on further study of the pathophysiology underlying this toxicity profile and make strides toward a more comprehensive assessment and treatment approach.

A tour de force provides novel insights into CD19-CAR T cell resistance mechanisms.

Sworder et al.1 developed an integrated simultaneous tumor and effector profiling (STEP) approach to study resistance mechanisms to CD19-CAR T cell therapy in large B-cell lymphomas. Their study provides novel biological insights and paves the way for future interventions.

Development of a cGMP-compliant process to manufacture donor-derived, CD45RA-depleted memory CD19-CAR T cells.

Autologous chimeric antigen receptor (CAR) T cells targeting the CD19 antigen have demonstrated a high complete response rate in relapsed/refractory B-cell malignancies. However, autologous CAR T cell therapy is not an option for all patients. Here we optimized conditions for clinical-grade manufacturing of allogeneic CD19-CAR T cells using CD45RA-depleted donor memory T cells (Tm) for a planned clinical trial. Tm were activated using the MACS GMP T Cell TransAct reagent and transduced in the presence of LentiBOOST with a clinical-grade lentiviral vector that encodes a 2nd generation CD19-CAR with a 41BB.zeta endodomain. Transduced T cells were transferred to a G-Rex cell culture device for expansion and harvested on day 7 or 8 for cryopreservation. The resulting CD19-CAR(Mem) T cells expanded on average 34.2-fold, and mean CAR expression was 45.5%. The majority of T cells were CD4+ and had a central memory or effector memory phenotype, and retained viral specificity. CD19-CAR(Mem) T cells recognized and killed CD19-positive target cells in vitro and had potent antitumor activity in an ALL xenograft model. Thus we have successfully developed a current good manufacturing practice-compliant process to manufacture donor-derived CD19-CAR(Mem) T cells. Our manufacturing process could be readily adapted for CAR(Mem) T cells targeting other antigens.

What is Next in Pediatric B-cell Precursor Acute Lymphoblastic Leukemia.

Cure rates now exceed 90% in many contemporary trials for children with B-cell acute lymphoblastic leukemia (ALL). However, treatment remains suboptimal and therapy is toxic for all patients. New treatment options potentially offer the chance to reduce both treatment resistance and toxicity. Here, we review recent advances in ALL diagnostics, chemotherapy, and immunotherapy. In addition to describing recently published results, we also attempt to project the impact of these new developments into the future to imagine what B-ALL therapy may look like in the next few years.

Common Trajectories of Highly Effective CD19-Specific CAR T Cells Identified by Endogenous T-cell Receptor Lineages.

Current chimeric antigen receptor-modified (CAR) T-cell products are evaluated in bulk, without assessing functional heterogeneity. We therefore generated a comprehensive single-cell gene expression and T-cell receptor (TCR) sequencing data set using pre- and postinfusion CD19-CAR T cells from blood and bone marrow samples of pediatric patients with B-cell acute lymphoblastic leukemia. We identified cytotoxic postinfusion cells with identical TCRs to a subset of preinfusion CAR T cells. These effector precursor cells exhibited a unique transcriptional profile compared with other preinfusion cells, corresponding to an unexpected surface phenotype (TIGIT+, CD62Llo, CD27-). Upon stimulation, these cells showed functional superiority and decreased expression of the exhaustion-associated transcription factor TOX. Collectively, these results demonstrate diverse effector potentials within preinfusion CAR T-cell products, which can be exploited for therapeutic applications. Furthermore, we provide an integrative experimental and analytic framework for elucidating the mechanisms underlying effector development in CAR T-cell products. SIGNIFICANCE: Utilizing clonal trajectories to define transcriptional potential, we find a unique signature of CAR T-cell effector precursors present in preinfusion cell products. Functional assessment of cells with this signature indicated early effector potential and resistance to exhaustion, consistent with postinfusion cellular patterns observed in patients. This article is highlighted in the In This Issue feature, p. 2007.

Chimeric Antigen Receptor T-cell Therapy: Current Status and Clinical Outcomes in Pediatric Hematologic Malignancies.

Chimeric antigen receptor T-cell (CART) therapy has transformed the treatment paradigm for pediatric patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), with complete remission rates in key pivotal CD19-CART trials ranging from 65% to 90%. Alongside this new therapy, new toxicity profiles and treatment limitations have emerged, necessitating toxicity consensus grading systems, cooperative group trials, and novel management approaches. This review highlights the results of key clinical trials of CART for pediatric hematologic malignancies, discusses the most common toxicities seen to date, and elucidates challenges, opportunities, and areas of active research to optimize this therapy.

Preferential expansion of CD8+ CD19-CAR T cells postinfusion and the role of disease burden on outcome in pediatric B-ALL.

T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.

Infectious Complications in Pediatric, Adolescent and Young Adult Patients Undergoing CD19-CAR T Cell Therapy.

CD19-specific chimeric antigen receptor (CAR) T cell therapy has changed the treatment paradigm for pediatric, adolescent and young adult (AYA) patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, data on the associated infectious disease challenges in this patient population are scarce. Knowledge of infections presenting during treatment, and associated risk factors, is critical for pediatric cellular therapy and infectious disease specialists as we seek to formulate effective anti-infective prophylaxis, infection monitoring schemas, and empiric therapy regimens. In this work we describe our institutional experience in a cohort of 38 pediatric and AYA patients with CD19-positive malignancy treated with lymphodepleting chemotherapy (fludarabine/cyclophosphamide) followed by a single infusion of CD19-CAR T cells (total infusions, n=39), including tisagenlecleucel (n=19; CD19/4-1BB) or on an institutional clinical trial (n=20; CD19/4-1BB; NCT03573700). We demonstrate that infections were common in the 90 days post CAR T cells, with 19 (50%) patients experiencing a total of 35 infections. Most of these (73.7%) occurred early post infusion (day 0 to 28; infection density of 2.36 per 100 patient days-at-risk) compared to late post infusion (day 29 to 90; infection density 0.98 per 100 patient days-at-risk), respectively. Bacterial infections were more frequent early after CAR T cell therapy, with a predominance of bacterial blood stream infections. Viral infections occurred throughout the post infusion period and included primarily systemic reactivations and gastrointestinal pathogens. Fungal infections were rare. Pre-infusion disease burden, intensity of bridging chemotherapy, lymphopenia post lymphodepleting chemotherapy/CAR T cell infusion and development of CAR-associated hemophagocytic lymphohistiocytosis (carHLH) were all significantly associated with either infection density or time to first infection post CAR T cell infusion. A subset of patients (n=6) had subsequent CAR T cell reinfusion and did not appear to have increased risk of infectious complications. Our experience highlights the risk of infections after CD19-CAR T cell therapy, and the need for continued investigation of infectious outcomes as we seek to improve surveillance, prophylaxis and treatment algorithms.

Sub-myeloablative Second Transplantations with Haploidentical Donors and Post-Transplant Cyclophosphamide have limited Anti-Leukemic Effects in Pediatric Patients.

Pediatric patients with high-risk hematologic malignancies who experience relapse after a prior allogeneic hematopoietic cell transplant (HCT) have an exceedingly poor prognosis. A second allogeneic HCT offers the potential for long-term cure but carries high risks of both subsequent relapse and HCT-related morbidity and mortality. Using haploidentical donors for HCT (haploHCT) can expand the donor pool and potentially enhance the graft-versus-leukemia effect but is accompanied by a risk of graft-versus-host disease (GVHD). The goal of this protocol was to intensify the antileukemia effect of haploHCT for pediatric patients with hematologic malignancies that relapsed after prior allogeneic HCT, while limiting regimen-associated toxicities. This phase II clinical trial evaluated a sub-myeloablative preparative regimen consisting of anti-thymocyte globulin, clofarabine, cytarabine, busulfan, and cyclophosphamide, in combination with plerixafor to sensitize leukemic blasts. Participants received a mobilized peripheral blood unmanipulated haploidentical donor graft with one dose of post-transplant cyclophosphamide as GVHD prophylaxis, followed by natural killer (NK) cell addback. Here we report the clinical outcomes and immune reconstitution of 17 participants treated on the study and 5 additional patients treated on similar single-patient treatment plans. Of the 22 participants analyzed, 12 (55%) had active disease at the time of HCT. The regimen provided robust immune reconstitution, with 21 participants (95%) experiencing neutrophil engraftment at a median of 14 days after HCT. In this high-risk population, the overall survival was 45% (95% confidence interval [CI], 24%-64%), with a 12-month event-free survival of 31% (95% CI, 14%-51%) and cumulative incidence of relapse at 12 months of 50% (95% CI, 27%-69%). Four participants (18%) remain in remission at >5 years follow-up. Expected HCT-related organ-specific toxicities were observed, and 13 participants (59%) experienced acute or chronic GVHD. This intensified but sub-myeloablative regimen, followed by a high-dose unmanipulated haploidentical graft, post-transplantation cyclophosphamide, and NK cell infusion, resulted in adequate immune reconstitution but failed to overcome the elevated risks of relapse and treatment-related morbidity in this high-risk population.