Abstract 3501: Induction of tumor cell apoptosis & immunogenic cell death by CD40L-armed oncolytic adenoviruses

Low tumor immunogenicity and a cold tumor microenvironment are two main characteristics occurring in different cancer types responsible for immunotherapy resistance. Immunostimulatory gene therapy using oncolytic viruses (OVs) has been developed as a new promising cancer therapy for its capacity to induce an anti-tumor immune response and sensitize the non-responding patients to checkpoint blockade therapy. Lokon adenoviruses (LOAds) are serotype 5/35 oncolytic adenoviruses that in addition to their oncolytic capacity encode for immunostimulatory molecules. Here we have evaluated the anti-tumor effects of trimerized membrane-bound (TMZ)-CD40L-armed LOAd700 and TMZ-CD40L/4-1BBL-armed LOAd703 in a variety of solid tumors to understand the underlying tumor modulating mechanisms. A panel of eight different human solid cancer cell lines (T24, PEA2, Mel526, H727, Skov3, MiaPaca2, H29 and A549) was infected with LOAd(-), a virus without transgenes, LOAd700 or LOAd703 and assessed for their response in different assays. MTS assay was used to measure the cell viability, flow cytometry for evaluating the cell surface markers and luminescent assay for measuring apoptosis (caspase 3/7 activity). LOAd viruses efficiently killed all tested cell lines. However, the oncolytic capacity of CD40L-armed LOAd700 and LOAd703 was significantly higher than LOAd(-) in two cell lines, T24 and PEA2, which were found to express the CD40 receptor. Death receptor TRAIL-R1 was significantly upregulated in T24 infected with LOAd703 and PEA2 infected with either LOAd700 or LOAd703. TRAIL-R2 was upregulated post LOAd infection in 7/8 cell lines where the upregulation was significant in T24, PEA2 and H29. Interestingly, the immunogenic cell death marker calreticulin, a damage-associated molecular pattern (DAMP) known as an “eat-me” signal to dendritic cells, was upregulated post LOAd infection in all cell lines. The increase in calreticulin level was statistically significant in 4/8 cell lines (T24, PEA2, Mel526 and H29) post LOAd700 and/or LOAd703 infection. Furthermore, LOAd700 and LOAd703 increased the effector caspase 3/7 activity in 5/8 cell lines (T24, PEA2, Mel526, Skov3 and MiaPaCa2) indicating an augmented apoptosis level. In conclusion, CD40L-armed LOAd viruses were, in addition to their high oncolytic capacity, demonstrated to induce apoptosis. Upregulation of cell surface calreticulin as well as the death receptors TRAIL-R1 and -R2 indicated generation of a more immunogenic phen