Use of anti-GD2 (Dinutuximab) as a target for CAR-T cells immunotherapy in neuroblastoma

Abstract

Treating solid tumours with chimeric antigen receptors-modified T cells (CAR-T cells) has shown limited efficacy due to the lack of cancer-type specific antigens. Dinutuximab is a monoclonal antibody that recognizes a sphingolipid, disialoganglioside GD2, which has limited expression in normal tissues but is overexpressed in paediatric tumours, mainly neuroblastoma (NB) and diffuse intrinsic pontine glioma (DIPG) and other histone H3 K27M (H3K27M) mutated diffuse midline gliomas. Dinutuximab, an anti-GD2 antibody, is currently standard of care in the treatment of NB. We are contemplating the possibility of using Dinutuximab as target element for antiNB-CAR-T cells. Our group have explored the feasibility of producing CAR-T cells from peripheral blood (PB), post-apheresis CD45RA+ fraction and cord blood (CB) using similar protocols of T cell transduction and expansion ability as a way to favour the persistence of CAR-T cells in the host. Afterward, we performed a deeply characterization of phenotype and functionality of anti-FITC-CAR-T cells derived from different sources, and the cytotoxic effect against anti-GD2-FITC- NB labelled cell line. T cells purified from PB, CB and 45RA fraction after apheresis were cultured in vitro with cytokines IL-7+IL-15+IL- 21 to maintain early memory phenotypes as Naïve T cells (TN). A second generation lentiviral expression vector (LV) was used to generate second generation CARs (FITC-L-BBz CAR). After 48h of activation in culture with antiCD3/CD28 and cytokines, T cells were transduced with viral supernatants. Next, we performed a characterization of phenotype of anti-FITC-CAR-T cells by flow cytometry studying how the culture conditions enhanced a TN phenotype expressing CD45RA+ and CCR7+. Cytotoxic function of CAR-T cells was analysed in cocultures with LAN-1 NB cell line labelled with Dinutuximab conjugated with FITC. Preliminary results showed that culture condition with IL-7/15/21 maintained more primitive phenotypes in 45RA (71% TN CD4+ and 66% TN CD8+) and CB (60% of TN CD4+ and 77% of TN CD8+) derived T cells than PB T cells (only a 20% of TN CD4+ and TN CD8+). PB and CB-derived CAR-T cells showed higher cytolitic function than 45RA fraction, resulting in a 30% of dead cells in both cases in a 24h-culture assay. Both, PB and CB derived CAR-T cells showed the best CAR-T activation capacity with 19,6% of cells from PB and 33,9% of cells from CB expressing early activation markers as CD25 and CD134. In vivo experiments are currently on-going to test the efficacy of CAR-T cells in combination with GD2 treatment. Our strategy may complement the current use of Dinutuximab in the treatment of NB through its combination with a targeted CAR-T cell approach.