Development of novel anti-cancer immunotherapies against advanced melanoma

Melanoma, an aggressive malignancy arising from melanocytes, remains a major therapeutic challenge at advanced stages, as conventional approaches—including surgery, chemotherapy, radiotherapy, and targeted therapy—often show limited efficacy. In this scenario, a game-changer has emerged: immunotherapy. Harnessing the patient’s own immune system, it has opened new horizons in the fight against melanoma. Among these approaches, immune checkpoint inhibitors (ICIs) block inhibitory pathways and restore the ability of T cells to fight tumours. ICIs appear to be an effective treatment for melanoma as they stimulate an immune response against cancer cells, eradicating the tumour. Yet, many patients remain non-responsive, which significantly limits their overall success. A promising strategy involves oncolytic viruses (OVs), which employ a dual mechanism of action: they directly destroy tumour cells while stimulating an antitumoral immune response. Still as monotherapy, their efficacy has proven to be modest, underscoring the need for combination strategies to unlock their full potential. Thus, the aim of this thesis project regards the development of a novel immunotherapeutic strategy for advanced melanoma combining third-generation oncolytic adenoviruses (OAds) with ICIs. We hypothesize that OAd-induced immunogenic cell death and remodelling of the tumour microenvironment (TME) will sensitize tumours to ICI therapy. The third-generation viruses — KUR05, KUR06, KUR07 and KUR08 — were engineered in collaboration with the National Institute of Public Health in Warsaw, Poland, and are armed with immunostimulatory transgenes to enhance T-cell recruitment, activation and remodelling of the tumour microenvironment. The experimental design focuses on evaluating both the efficacy and safety of these novel viruses in preclinical melanoma models. Two complementary in vitro systems have been employed: 2D cell cultures to assess key mechanistic parameters, and 3D culture based spheroid models that better recapitulate the immunosuppressive TME and incorporate an immune component through peripheral blood mononuclear cell (PBMC) co-culture. Physicochemical characterisation confirmed that all viral candidates were the appropriate size and exhibited the necessary stability and surface charge. Receptor profiling indicated that melanoma cell lines expressed the Ad5/3 entry receptors DSG-2 and CD46, which supports efficient infection. In 2D assays, all oncolytic adenoviruses (OAds) reduced cell viability in a dose-dependent manner and consistently induced immunogenic cell death (ICD). As expected, ICIs alone or in combination had limited efficacy in 2D due to the absence of immune components. In contrast, the 3D spheroid–peripheral blood mononuclear cell (PBMC) co-culture models provided a more informative context for evaluating immune-dependent activity. In this setting, all of the oncolytic adenoviruses that were tested led to a progressive reduction in spheroid size, which confirmed their anti-tumour efficacy in a structurally and immunologically enriched environment. Combining OAds with ICIs produced a further, albeit modest, improvement over viral monotherapy, with the most pronounced response being observed in A375 spheroids. As expected, ICIs alone had a minimal impact on both analysed models. Overall, these results suggest that the newly generated OAds retain anti-tumour activity in 3D systems and that the presence of immune cells is necessary to observe the effects of the combined treatment.