The Italian researchers enroled in an academic, phase I/II study patients who are 1 to 25 years of age with relapsed or refractory, high-risk neuroblastoma to test autologous, third-generation GD2-CAR T cells expressing the inducible caspase 9 suicide gene (GD2-CART01). The study showed that GD2-CART01 cells expanded to high levels in vivo. These cells also induced an immune-related response that was manifested in part by mild cytokine release syndrome (CRS) in most of the patients and by sustained clinical responses, with a complete response in 33% of the patients. Among the patients who received the recommended dose, the 3-year overall survival (OS) and event-free survival (EFS) were 60% and 36%, respectively.

The findings are published by Dr. Franco Locatelli of the Department of Pediatric Hematology and Oncology and of Cell and Gene Therapy, IRCCS Ospedale Pediatrico Bambino Gesù in Rome, Italy and colleagues on 6 April 2023 in The New England Journal of Medicine. They wrote that the use of GD2-CART01 was feasible and safe in patients with high-risk neuroblastoma. Treatment-related side effects developed, and the activation of the suicide gene controlled them. GD2-CART01 may have a sustained antitumor effect in patients with relapsed or refractory neuroblastoma. Studies are under way to assess the role of GD2-CART01 in the multimodal treatment of high-risk neuroblastoma.

The authors wrote in the background that neuroblastoma cells express high levels of the disialoganglioside GD2, and the targeting of disialoganglioside GD2 with monoclonal antibodies has been associated with a significant increase in survival among patients with high-risk disease, findings that indicate the sensitivity of neuroblastoma to immunotherapy and the relevance of the target antigen. Few early-phase clinical studies of GD2-targeted CAR T cells have shown that the approach is feasible, and few objective responses have been reported.

The study team developed a GD2-directed CAR construct incorporating two costimulatory domains — CD28 and 4-1BB. To address potential neurotoxic effects associated with the use of GD2-CART01, third-generation CAR T cells, the study team also included in the construct the gene for inducible caspase 9, as a safety switch allowing the adoptively transferred cells to be killed if they were associated with dangerous side effects.

A total of 27 patients with heavily pretreated neuroblastoma, of whom 12 with refractory disease, 14 with relapsed disease, and 1 with a complete response at the end of first-line treatment, were enroled and received GD2-CART01. No failure to generate GD2-CART01 was observed. Three dose levels were tested, 3x, 6x, and 10×106 CAR-positive T cells per kilogram of body weight in the phase I portion of the study, and no dose-limiting side effects were recorded. The recommended dose for the phase 2 portion of the study was 10×106 CAR-positive T cells per kilogram.

CRS occurred in 20 of 27 patients (74%) and was mild in 19 of 20 (95%). In 1 patient, the suicide gene was activated, with rapid elimination of GD2-CART01. GD2-targeted CAR T cells expanded in vivo and were detectable in peripheral blood in 26 of 27 patients up to 30 months after infusion with median persistence of 3 months (range, 1 to 30).

A total, 17 patients had a response to the treatment; overall response was 63%, 9 patients had a complete response, and 8 had a partial response. Among the patients who received the recommended dose, the 3-year OS and EFS were 60% and 36%, respectively.

To maintain tumour control, it is essential that CAR T cells persist over time. In contrast to patients in previous studies, all patients in this study had persistence of CAR T cells for more than 6 weeks, possibly because of the incorporation of two costimulatory domains in the construct and the use of interleukin-7 and interleukin-15 in the manufacturing of GD2-CART01.

The authors commented that although the study was not powered to detect differences between subgroups of patients, they identified disease burden at infusion as being predictive of response to treatment. They did not observe a difference in response and in long-term outcomes between patients with refractory disease and those with relapsed disease, and the previous use of anti-GD2 monoclonal antibodies did not influence outcomes.

In contrast to observations with other target antigens such as CD19, CD22, and GPC2, they did not observe a loss of GD2 expression on relapsed tumour cells after loss of response to the treatment, as shown by the mean fluorescence intensity of the antigen at relapse. These findings, coupled with the lack of re-expansion of GD2-CART01 on relapse or progression of the disease, suggest that in neuroblastoma, mechanisms of resistance other than antigen loss and short persistence of CAR T cells are responsible for treatment failure.

In an accompanied editorial article, Drs. Oladapo O. Yeku and Dan L. Longo of the Mass General Cancer Center and Harvard Medical School Boston, MA, US wrote that in this study the tumours in patients who did not have a response retained expression of GD2, a finding that raises the question of whether immunosuppressive cytokines, myeloid-derived suppressor cells, T-regulatory cells, or tumour-associated macrophages contribute to a lack of efficacy in these patients. The metabolic fitness of the CAR T effector cells derived in each patient is also unknown and could inform cytolytic capacity and susceptibility to dysfunction. The extent to which the patients’ tumours maintained an immunosuppressive environment and whether this affected the efficacy of autologous GD2-CART01 is not known. A nuanced understanding of these and other potential mechanisms of treatment failure may set the stage for tailored and rationally designed combination treatments in the future.

The study was supported by grants from the Italian Medicines Agency–Ricerca Indipendente, the Italian Ministry of Health, Associazione Italiana per la Ricerca sul Cancro (AIRC), Innovative Medicines Initiative 2 Joint Undertaking, Lazio Innova Project Immunotherapy Approaches, project CAR for Sarcoma, Fondazione Neuroblastoma, Italian Piano Nazionale di Ripresa e Resilienza CN3, and Life-Science Hub–Terapie Avanzate. Bellicum Pharmaceuticals donated rimiducid for the study.

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