Modulating Oncolytic Adenovirus Immunotherapy by Driving Two Axes of the Immune System by Expressing 4-1BBL and CD40L

Oncolytic viruses (OVs) can have utility for direct killing of cancer cells, but may also serve to activate the immune system against cancer cells. While viruses alone can serve as immune stimulators, there is great interest in arming OVs with genes encoding immune stimulatory proteins to amplify their effects. In this work, we have tested the efficacy of conditionally-replicating adenoviruses (CRAds) with and without selected immunostimulatory payloads, murine CD40L (mCD40L) or 4-1BBL (m4-1BBL), in an immune competent mouse model of melanoma. When CRAd657-m4-1BBL and CRAd657-mCD40L were injected into B16-hCAR murine melanoma tumors, both single vectors delayed tumor growth and prolong survival compared to empty CRAd657. However, combined injection of both CRAd-m4-1BBL and CRAd-mCD40L mediated significantly better control of tumor growth. All of the payloads increased immune cell infiltration into tumors and notably reduced expression of PD-1 exhaustion marker on T cells. Tumor volumes were negatively associated with total infiltrating T cell population. We found that the payloads increased immune cell infiltration into tumors with some specificities: recruitment of CD8⁺ T cells was higher with m4-1BBL expression, while mCD40L expression induced more CD4⁺ T cell infiltration. Importantly, the combination of CRAd657-m4-1BBL and CRAd657-mCD40L induced the highest immune cells/T cell infiltration and the highest anti-TRP-2 tumor-associated antigen T cell responses than empty or single gene vector. This combination also caused depigmentation in areas adjacent to the tumor sites in more animals. These data indicate that driving two axes of the immune system with combined immune stimulatory payloads can lead to improved anticancer immune responses and better tumor control in an immune competent model of cancer.