Vitale, Maria (2022) ONCOLYTIC ADENOVIRAL VECTOR-MEDIATED EXPRESSION OF AN ANTI-PD-L1 SCFV IMPROVES ANTI-TUMORAL EFFICACY IN A MELANOMA MOUSE MODEL. [Tesi di dottorato]

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Oncolytic virotherapy is an emerging therapeutic approach, based on replication-competent viruses able to selectively infect and destroy cancer cells causing the release of tumor-associated antigens, therefore stimulating an antitumoral immune response. Indeed, adenoviruses (Ads), especially, oncolytic adenoviruses (Onc. Ads), can kill cancer cells in different ways by inducing immunogenic cell death. To increase their anticancer activity we engineered the Ad5Δ24 to add the ability to block tumor immune evasion. Programmed death ligand 1 (PD-L1) binding to its receptor PD-1 inhibits CD8+ T cell proliferation turning off the antitumoral T cell-mediated response. Therefore, we isolated a single-chain variable antibody fragment (scFv) that binds to both human and murine PD-L1 with high affinity and developed an Onc.AdΔ24 (Ad5Δ24-scFv-anti-PD-L1) that expresses the scFv to combine blockage of PD-1/PD-L1 interaction with the antitumoral activity of Onc.Ads. To assess the efficacy of the modified Ads, we infected a human melanoma cell line, SK-MEL 28, with different Multiplicity Of Infection (MOI) of Ad5Δ24-scFv-anti-PD-L1. At 48 hours post-infection, we evaluated cell death, and observed a decrease of cell survival at higher MOI of the virus, indicating a dose-response correlation between cell death and MOI. These data suggest that scFv expression enhanced the oncolytic activity of Ad, mediating a robust effect on cancer cell survival. Furthermore, we tested the Ad5Δ24-scFv-anti-PD-L1, on B16.OVA cells, a murine model of melanoma. We infected B16.OVA and then co-cultured with C57BL/6 naïve splenocytes, to mimic a simplified immune system, in vitro. We observed that the treatment with the Ad5Δ24-scFv-anti-PD-L1 induced a significantly higher cancer cell death. Furthermore, we administered our new Ads intratumorally in C57BL/6 mice engrafted with B16.OVA and followed tumor progression compared to Ad5Δ24-scFv-anti-PD-L1 a control group treated with the un-modified Onc.Ad. We observed that treatment induced a reduction of tumor growth compared to controls and in addition, the evaluation of the lymphocytic population infiltrating the tumor reveal a favorable immune profile with an enhancement of the CD8+ population. These data suggest, that Onc.Ad-induced expression of an immune checkpoint inhibitor is an effective and promising strategy in cancer treatments, opening a new way into cancer therapy.

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