Gene Therapy for Neuroblastoma: Preclinical Evaluation of a Third Generation Chimeric Antigen Receptor Directed Against GD2

Neuroblastoma (NB) is a tumor derived from the sympathetic nervous system that accounts for 8-10% of childhood cancers and it is the most common malignant extracranial solid tumor in the pediatric population. Although therapeutic approaches based on chemo/radiotherapy and surgery can induce remission in the majority of patients with low/intermediate risk disease, more than 60% of patients with high-risk NB exhibit a poor prognosis, despite intensive therapy. Therefore novel curative approaches are urgently needed to improve the outcome of these patients. Advanced NB appears susceptible to immunotherapies, including monoclonal antibodies and T-cell based strategies. To combine beneficial effects of both humoral and T-cell mediated immune response, autologous T lymphocytes have been genetically modified to express chimeric protein known as chimeric antigen receptors (CARs), that combine the antigen binding specificity of a monoclonal antibody with the effector endodomain of the CD3/T cell receptor complex (ζchain). In particular, a CAR targeting the disialoganglioside GD2 (CAR-GD2), an antigen expressed by the great majority of NB cells and poorly expressed on normal tissues, have been validated in the preclinical and clinical setting, showing promising results for the treatment of patients affected by relapsed/refractory NB. In order to increase the efficacy of CAR.GD2 based therapy, a possible strategy is represented by the insertion in the CAR.GD2 construct itself of one or more costimulatory endodomains (second-third generation CARs), in order to allow, after CAR engagement, appropriate T-cell activation, that is necessary to ensure long-term proliferation, persistence and anti-tumor effect. Moreover, the safety of this approach can be implemented by the addition of a safety switch based on an inducible suicide gene that allows the rapid and selective ablation of the cells expressing the construct in the presence of unexpected toxicity. Based on this rationale, the aim of this project is to assess in vitro and in vivo the proliferation and the antitumor efficacy of T cells expressing a third generation GD2-CAR which incorporates OX40 costimulatory molecule and a safety switch.