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

Oncolytic viruses 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 oncolytic viruses with the genes for immune stimulatory proteins to amplify their effects. In this work, we have tested the efficacy of a conditionally-replicating adenoviruses (CRAds) with and without selected immunostimulatory payloads in an immune competent mouse model of melanoma. Empty CRAd657 was compared to the same vector expressing mouse CD40L or mouse 4-1BBL. When CRAd657-m4-1BBL and CRAd657-mCD40L were injected into B16-hCAR murine melanoma tumors, both single vectors delayed tumor growth and prolong survival when compared to empty CRAd657. However, combined injection of both CRAd-4-1BBL and CRAd-CD40L 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. However, recruitment of CD8+ T cells was higher with 4-1BBL alone while CD40L expression induced more CD4+ T cell infiltration. Notably, the combination of CRAd657-4-1BBL and CRAd657-CD40L induced higher anti-TRP-2 tumor-associated antigen T cell responses than empty or single gene vectors. 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 anti-cancer immune responses and better tumor control in an immune competent model of cancer.