Abstract
Pediatric high-grade gliomas (pHGG) are amongst the most common malignant neoplasms of childhood, whose outcome remains dismal with conventional treatments. New therapeutic approaches are urgently needed. Immunotherapy based on Oncolytic Adenovirus(OA)is a promising strategy but its efficacy is suboptimal. We aimed at improving the antitumor efficacy by combining the OA and a gene-therapy with the Bibispecific T-cell Engager (BiTE) directed towards the erythropoietin-producing human hepatocellular carcinoma A2-receptor (EphA2),conveyed by a replication-incompetent adenoviral vector (EAd). We demonstrated, by immunohistochemistry and qPCR, the expression of EphA2 in 100% (16/16) of the pHGGs, its intensity correlating significantly with a worst outcome. We then tested the transgene amplification, after co-infection, on two HGG cell lines (U373, U87) by qPCR and Flow Cytometry (FACS) for the selectable marker ∆CD19, confirming a significantly enhanced production in OA+EAd vs EAd alone (p<0.01 and p<0.001, respectively). Notably, the FACS analysis of the infected tumor cells after 5 days of co-culture with T-cells showed a significantly increased apoptosis in the OA+EAd compared to the controls (p<0.001). To obtain the tumor eradication, we then fully activated the T-cells by including an anti-CD28 antibody (αCD28), revealing a further apoptosis enhancement. We then characterized the T-cell populations by FACS analysis showing a significant increase of activation and non-exhaustion markers and the addition of the αCD28 was able to induce a significantly higher production of interferon-γ in EAd or OA+EAd conditions. Finally, we established an orthotopic HGG mouse model and administered OA/Ead/OA+EAd and T cells intracranially. Preliminary data show that the treatment is well tolerated and that the EphA2-BiTE improves tumor control compared to the control groups. In conclusion, the combinatorial approach is able to amplify the production of the BiTE and to determine an effective tumor control in the presence of T-cells and αCD28, representing a promising innovative treatment.