Diffuse intrinsic pontine glioma (DIPG) is a fatal central nervous system (CNS) tumor that confers a median survival of 11 months. As B7-H3 is expressed on pediatric CNS tumors, we conducted BrainChild-03, a single-center, dose-escalation phase 1 clinical trial of repetitive intracerebroventricular (ICV) dosing of B7-H3-targeting chimeric antigen receptor T cells (B7-H3 CAR T cells) for children with recurrent or refractory CNS tumors and DIPG. Here we report results from Arm C, restricted to patients with DIPG. The primary objectives were to assess feasibility and tolerability, which were both met. Secondary objectives included assessments of CAR T cell distribution and survival. A total of 23 patients with DIPG enrolled, and 21 were treated with repeated doses of ICV B7-H3 CAR T cells using intra-patient dose-escalation regimens without previous lymphodepletion. Concurrent tumor-directed therapy, including re-irradiation, was not allowed while on protocol therapy. We delivered a total of 253 ICV doses and established the highest planned dose regimen, DR4, which escalated up to 10 × 107 cells per dose, as the maximally tolerated dose regimen. Common adverse events included headache, fatigue and fever. There was one dose-limiting toxicity (intratumoral hemorrhage) during DR2. For all treated patients (n = 21), the median survival from their initial CAR T cell infusion was 10.7 months and the median survival from diagnosis was 19.8 months with 3 patients still alive at 44, 45 and 52 months from diagnosis. Ultimately, this completed first-in-human trial shows that repetitive ICV dosing of B7-H3 CAR T cells in pediatric and young adult patients with DIPG is tolerable, including multiyear repeated dosing, and may have clinical efficacy that warrants further investigation on a multisite phase 2 trial. ClinicalTrials.gov registration: NCT04185038 . In the final report of a phase 1 trial evaluating intracerebroventricular B7-H3-targeting CAR T cells in children and young adults with diffuse intrinsic pontine glioma, repeated intracranial infusions were feasible and well tolerated with a median overall survival of 19.8 months and 3 patients surviving over 40 months from diagnosis.

Abstract Outcomes for children with relapsed and refractory neuroblastoma are dismal. ENCIT-01 is our first-in-human experience in patients with relapsed and refractory neuroblastoma using chimeric antigen receptor (CAR) T cells targeting L1-CAM, an adhesion molecule that is overexpressed in neuroblastoma with limited normal tissue expression. This trial evaluated three different CAR constructs: a short spacer second-generation 4-1BB CAR (Arm A), a short spacer third-generation 4-1BB+CD28 CAR (Arm B) and a long spacer second-generation 4-1BB CAR (Arm C). Thirty-six patients were enrolled and 22 were treated (Arm A n=11, Arm B n=8 and Arm C n=3). Cytokine-release syndrome, skin rash and dose-limiting hyponatremia were recurrently encountered toxicities. Patterns of toxicity appeared at lower dose levels on Arm B and Arm C compared to Arm A, suggesting enhanced potency of the third generation and long spacer products. No objective responses were seen. Correlative analyses demonstrated CAR T cells infiltration into tumor and skin, with evidence of macrophage tumor infiltration. In addition, enhanced CD107a production in the third-generation products when compared to patient matched second generation product, potentially explaining the observation of toxicities at lower dose levels. While feasible to manufacture in a heavily pretreated population, additional engineering safety of L1CAM CAR T cells and/or strategies to target the immunosuppressive tumor microenvironment may be needed to prevent toxicity and provide durable anti-tumor effects.

BACKGROUND:A major obstacle in translating the therapeutic potential of chimeric antigen receptor (CAR) T cells to children with central nervous system (CNS) tumors is the blood-brain barrier. To overcome this limitation, preclinical and clinical studies have supported the use of repeated, locoregional intracranial CAR T-cell delivery. However, there is limited literature available describing the process for the involvement of an investigational drug service (IDS) pharmacy, particularly in the setting of a children's hospital with outpatient dosing for CNS tumors. OBJECTIVES:To describe Seattle Children's Hospital's experience in clinically producing CAR T cells and the implementation of IDS pharmacy practices used to deliver more than 300 intracranial CAR T-cell doses to children, as well as to share how we refined the processing techniques from CAR T-cell generation to the thawing of fractionated doses for intracranial delivery. METHODS:Autologous CD4+ and CD8+ T cells were collected and transduced to express HER2, EGFR, or B7-H3-specific CAR T cells. Cryopreserved CAR T cells were thawed by the IDS pharmacy before intracranial delivery to patients with recurrent/refractory CNS tumors or with diffuse intrinsic pontine glioma/diffuse midline glioma. RESULTS:The use of a thaw-and-dilute procedure for cryopreserved individual CAR T-cell doses provides reliable viability and is more efficient than typical thaw-and-wash protocols. Cell viability with the thaw-and-dilute protocol was approximately 75% and was always within 10% of the viability assessed at cryopreservation. Cell viability was preserved through 6 hours after thawing, which exceeded the 1-hour time frame from thawing to infusion. CONCLUSION:As the field of adoptive immunotherapy grows and continues to bring hope to patients with fatal CNS malignancies, it is critical to focus on improving the preparatory steps for CAR T-cell delivery.

Diffuse intrinsic pontine glioma (DIPG) is a fatal central nervous system (CNS) tumor with a median survival of 11 months. Chimeric antigen receptor (CAR) T cells are clinically effective against hematologic malignancies, but there are limited data on their utility against CNS tumors. As B7-H3 is expressed on pediatric CNS tumors, we conducted BrainChild-03 (NCT04185038), a single-center, dose-escalation phase 1 study of repetitive intracerebroventricular (ICV) B7-H3 CAR T cells for children with recurrent/refractory CNS tumors and DIPG. Here, we report on Arm C restricted to patients with DIPG. The primary objectives were to assess feasibility and tolerability. The secondary objectives were to assess CAR T cell distribution and overall survival. Twenty-three patients with DIPG enrolled and 21 were treated with ICV B7-H3 CAR T cells using intra-patient dose escalation dose regimens (DR) beginning at 1x107 CAR T cells/dose. We established the DR4, the highest planned DR that escalated to 10x107 cells/dose, as the maximally tolerated dose regimen (MTDR). Common adverse events included fever and headache. There was 1 DLT (intratumoral hemorrhage). An intent-to-treat analysis of median overall survival of enrolled patients (n = 23) from study enrollment was 11.4 months (range: 2.7–36.8) and the median time from initial diagnosis to death (or last contact for survivors) was 18.6 months (range: 6.5–40.5) with 4 patients still alive at 18.6, 37.2, 38.3, 40.5 months from diagnosis. Ultimately, the report of this completed first-in-human trial demonstrates that repetitive ICV B7-H3 CAR T cells in pediatric and young adult patients with DIPG is tolerable, even over repeated multi-year dosing, and may improve overall survival. Main Text

Supplementary Data from Modified Manufacturing Process Modulates CD19CAR T-cell Engraftment Fitness and Leukemia-Free Survival in Pediatric and Young Adult Subjects

Central nervous system (CNS) tumors are the most common solid malignancy in the pediatric population. Based on adoptive cellular therapy's clinical success against childhood leukemia and the preclinical efficacy against pediatric CNS tumors, chimeric antigen receptor (CAR) T cells offer hope of improving outcomes for recurrent tumors and universally fatal diseases such as diffuse intrinsic pontine glioma (DIPG). However, a major obstacle for tumors of the brain and spine is ineffective T cell chemotaxis to disease sites. Locoregional CAR T cell delivery via infusion through an intracranial catheter is currently under study in multiple early phase clinical trials. Here, we describe the Seattle Children's single-institution experience including the multidisciplinary process for the preparation of successful, repetitive intracranial T cell infusion for children and the catheter-related safety of our 307 intracranial CAR T cell doses.

AbstractT cells modified to express a chimeric antigen receptor (CAR) targeting CD19 can induce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). The durability of remission is related to the length of time the CAR T cells persist. Efforts to understand differences in persistence have focused on the CAR construct, in particular the costimulatory signaling module of the chimeric receptor. We previously reported a robust intent-to-treat product manufacturing success rate and remission induction rate in children and young adults with recurrent/refractory B-ALL using the SCRI-CAR19v1 product, a second-generation CD19-specific CAR with 4-1BB costimulation coexpressed with the EGFRt cell-surface tag (NCT02028455). Following completion of the phase I study, two changes to CAR T-cell manufacturing were introduced: switching the T-cell activation reagent and omitting midculture EGFRt immunomagnetic selection. We tested the modified manufacturing process and resulting product, designated SCRI-CAR19v2, in a cohort of 21 subjects on the phase II arm of the trial. Here, we describe the unanticipated enhancement in product performance resulting in prolonged persistence and B-cell aplasia and improved leukemia-free survival with SCRI-CAR19v2 as compared with SCRI-CAR19v1.

Chimeric antigen receptor (CAR) T cell therapy provides a broadly applicable, targeted, yet pathway-independent, intervention for pediatric central nervous system (CNS) tumors. We have optimized the efficacy and specificity of a HER2-targeted CAR with an extracellular target-specific scFv domain derived from trastuzumab (a HER2 targeting antibody), a co-expressed truncated EGFR (EGFRt) to evaluate transduction efficacy and as a potential suicide mechanism with cetuximab treatment, and a medium-length spacer coupled to an intracellular 4-1BBζ domain. In vitro studies revealed antigen specific cytotoxicity and cytokine release against HER2-positive CNS tumors; and the HER2CAR significantly improved survival (p=0.003) in vivo at 90 days in a xenograft CNS model relative to Mock-treated controls. BrainChild-01 is a first-in-human trial evaluating locoregional infusion of HER2CAR T cells for children and young adults with HER2-positive progressive or recurrent CNS tumors. Autologous T cells are apheresed, engineered to express the second-generation HER2CAR, and expanded ex vivo. They are delivered via indwelling CNS catheter into either the tumor cavity (Arm A) or ventricular system (Arm B) using an intra-patient dose-escalation regimen with weekly dosing for 3 of every 4 weeks per course. The study period is two courses though subjects may receive up to six. Subject 001, a 19-year-old female with a parietal lobe hypermutated HER2-positive anaplastic astrocytoma (WHO grade III), enrolled on Arm A following third progression, underwent apheresis and successful production of 1.9 x 109 HER2CAR T cells. She tolerated the study-prescribed six CAR T cell doses over two courses at doses of 1 x 107 to 2.5 x 107 without dose limiting toxicity. Acute local inflammation and CRP elevation followed each dose. MRI brain/spine obtained after Course 2 revealed increased enhancement and T2/FLAIR hyperintensity surrounding the tumor cavity with mildly increased mass effect, indeterminate for treatment-related inflammatory changes/pseudoprogression versus tumor progression. Enrollment is ongoing.

Cytotoxic chemotherapy and radiation can render lymphocyte repertoires qualitatively and quantitatively defective. Thus, heavily treated patients are often poor candidates for the manufacture of autologous chimeric antigen receptor (CAR)-T cell products. In the United States and Europe, children with high-risk neuroblastoma undergo apheresis early in the course of treatment to collect peripheral blood stem cells (PBSCs) for cryopreservation in preparation for high-dose chemotherapy followed by autologous stem cell rescue. Here, we investigate whether these cryopreserved chemotherapy and granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs can serve as starting material for CAR-T cell manufacturing. We evaluated T cell precursor subsets in cryopreserved PBSC units from 8 patients with neuroblastoma using fluorescent activated cell sorting-based analysis. Every cryopreserved unit collected early in treatment contained both CD4 and CD8 precursors with significant numbers of na ve and central memory precursors. Significant numbers of Ki67(+)/PD1(+) T cells were detected, presumably the result of chemotherapy-induced lymphopenia and subsequent homeostatic proliferation. Cryopreserved PBSC units containing 56 to 112 x 10(6) T cells were amenable to immunomagnetic selection, CD3 x 28 bead activation, lentiviral transduction, and cytokine-driven expansion, provided that CD14 monocytes were depleted before the initiation of cultures. Second- and third-generation CD171 CAR(+) CD4 and CD8 effector cells derived from cryopreserved units displayed antineuroblastoma lytic potency and cytokine secretion comparable to those derived from a healthy donor and mediated in vivo antitumor regression in NSG mice. We conclude that cryopreserved PBSCs procured via standard methods during early treatment can serve as an alternative starting source for CAR-T cell manufacturing, extending the options for heavily treated patients. (C) 2018 American Society for Blood and Marrow Transplantation.