Purpose: Medulloblastoma (MB), the most common childhood malignant brain tumor, has a poor prognosis in about 30% of patients. The current standard of care, which includes surgery, radiation, and chemotherapy, is often responsible for cognitive, neurologic, and endocrine side effects. We investigated whether chimeric antigen receptor (CAR) T cells directed toward the disialoganglioside GD2 can represent a potentially more effective treatment with reduced long-term side effects. Experimental Design: GD2 expression was evaluated on primary tumor biopsies of MB children by flow cytometry. GD2 expression in MB cells was also evaluated in response to an EZH2 inhibitor (tazemetostat). In in vitro and in vivo models, GD2+ MB cells were targeted by a CAR-GD2.CD28.4-1BBζ (CAR.GD2)-T construct, including the suicide gene inducible caspase-9. Results: GD2 was expressed in 82.68% of MB tumors. The SHH and G3–G4 subtypes expressed the highest levels of GD2, whereas the WNT subtype expressed the lowest. In in vitro coculture assays, CAR.GD2 T cells were able to kill GD2+ MB cells. Pretreatment with tazemetostat upregulated GD2 expression, sensitizing GD2dimMB cells to CAR.GD2 T cells cytotoxic activity. In orthotopic mouse models of MB, intravenously injected CAR.GD2 T cells significantly controlled tumor growth, prolonging the overall survival of treated mice. Moreover, the dimerizing drug AP1903 was able to cross the murine blood–brain barrier and to eliminate both blood-circulating and tumor-infiltrating CAR.GD2 T cells. Conclusions: Our experimental data indicate the potential efficacy of CAR.GD2 T-cell therapy. A phase I/II clinical trial is ongoing in our center (NCT05298995) to evaluate the safety and therapeutic efficacy of CAR.GD2 therapy in high-risk MB patients.

Autologous CD19-directed chimeric antigen receptor (CAR)-T cells have shown unprecedented efficacy in children with relapsed/refractory B-cell-precursor acute lymphoblastic leukemia (BCP-ALL). However, patients either relapsing after allogeneic hematopoietic stem cell transplantation (allo-HSCT), or displaying profound lymphopenia and/or with rapidly progressing disease often cannot access autologous products. These hurdles may be overcome by allogeneic, donor-derived CAR-T cells. We tested donor-derived T-cells transduced with a 2nd-generation (4.1BB) CD19-CAR for treatment of patients with BCP-ALL, in a hospital exemption setting. Two constructs were tested: a retroviral construct incorporating the suicide gene inducible caspase-9 (CD19-CAR-Retro_ALLO) first and then a lentiviral construct and an automated, Prodigy®-based, manufacturing process (CD19-CAR-Lenti_ALLO). Thirteen children/young adults received ALLO-CAR T-cells between 03/2021 and 10/2022. Doses ranged between 1,0×106 and 3,0×106 CAR T-cells/kg. The toxicity profile was comparable to that of autologous CAR-T cells, characterized mainly by cytopenia, CRS (maximum grade 1) and grade 2 ICANS. One case of acute graft-versus-host disease (GvHD) occurred and was rapidly controlled by steroids and ruxolitinib. None of the other patients, including 3 infused with ALLO-CAR T cells from an HLA-haplo-identical donor, experienced GvHD. Two patients received ALLO-CAR T-cells before HSCT and showed a significant expansion of CAR T cells, without any sign of GvHD. All patients obtained complete remission (CR) with negativity of minimal residual disease in the BM; with a median follow-up of 12 months (range 5-21), 8/13 patients maintain CR. Allogeneic anti-CD19 CAR-T cells can effectively treat highly-refractory BCP-ALL relapsing after alloHSCT, without showing increased toxicity as compared to autologous CAR T cells.

TCRαβ/CD19 cell depletion is a promising graft manipulation technique frequently used in the context of human leukocyte antigen (HLA)–haploidentical hematopoietic stem cell transplantation (HSCT). We previously reported the results of a phase I-II clinical trial (NCT01810120) to assess the safety and the efficacy of this type of exvivo T-cell depletion in 80 children with acute leukemia, showing promising survival outcomes. We now report an updated analysis on a cohort of 213 children with a longer follow-up (median, 47.6 months for surviving patients). With a 5-year cumulative incidence of nonrelapse mortality of 5.2% (95% confidence interval [CI], 2.8%-8.8%) and a cumulative incidence of relapse of 22.7% (95% CI, 16.9%-29.2%), projected 10-year overall and disease-free survival (DFS) were 75.4% (95% CI, 68.6%-80.9%) and 71.6% (95% CI, 64.4%-77.6%), respectively. Cumulative incidence of both grade II-IV acute and chronic graft-versus-host disease were low (14.7% and 8.1%, respectively). In a multivariable analysis for DFS including type of disease, use of total body irradiation in the conditioning regimen (hazard ratio [HR], 0.5; 95% CI, 0.26-0.98; P = .04), disease status at HSCT (complete remission [CR] ≥3 vs CR 1/2; HR, 2.23; 95% CI, 1.20-4.16; P = .01), and high levels of pre-HSCT minimal residual disease (HR, 2.09; 95% CI, 1.01-4.33; P = .04) were independently associated with outcome. In summary, besides confirming the good outcome results already reported (which are almost superimposable on those of transplant from HLA-matched donors), this clinical update allows the identification of patients at higher risk of treatment failure for whom personalized approaches, aimed at reducing the risk of relapse, are warranted.

The point-of-care, automated manufacturing of CAR T cells can reduce the waiting window and increase the therapy accessibility to patients with relapsed/refractory (r/r) BCP-ALL as compared to commercially available drug products (DP). We conducted a phase I/II trial (NCT04787263) on the use of 2nd generation (4.1BB), CD19-CAR T cells, for the treatment of BCP-ALL patients with either 1st relapse and very high-risk (VHR) characteristics or >2nd relapse and rapidly progressing disease preventing access to the commercial DP. CAR-T cells were manufactured with a 12-day process using CliniMACS Prodigy®, from a fresh apheresis and infused fresh. Three dose levels were tested in the phase I, namely 1.0, 2.0 and 3.0×106 CAR+T cells/kg. Nineteen pts were enrolled, 13/19 (68%) in first VHR relapse. The designed dose was always successfully produced. No dose-limiting toxicities were observed in the phase I. Grade 1-2 cytokine release syndrome was observed in 13/19 (68%) patients. Grade 1-2 immune effector cell-associated neurotoxicity syndrome occurred in 2 patients and resolved spontaneously. All patients achieved bone marrow complete remission with negative minimal residual disease. Eight pts (42%) received hematopoietic stem cell transplantation (HSCT) consolidation. Overall, CR was maintained in 13/19 patients (68%), 7 of them having received HSCT. Importantly, all patients but one treated with 3.0x106 CAR+T cells/kg maintain CR. The 3y-EFS and OS of the whole cohort are 68% and 83%, respectively. Our data suggest that the point-of-care, manufacturing of autologous, anti-CD19 CAR T cells can successfully treat patients with BCP-ALL including those with 1st relapse and VHR characteristics. (NCT04787263)