CD40 stimulation via CD40 ligand enhances adenovirus‐mediated tumour immunogenicity including ‘find‐me’, ‘eat‐me’, and ‘kill‐me’ signalling

Abstract Immunostimulatory gene therapy using oncolytic viruses is currently evaluated as a promising therapy for cancer aiming to induce anti‐tumour immunity. Here, we investigate the capacity of oncolytic adenoviruses (LOAd) and their transgenes to induce immunogenicity in the infected tumour cells. Oncolysis and death‐related markers were assessed after infection of eight human solid cancer cell lines with different LOAd viruses expressing a trimerized, membrane‐bound (TMZ)‐CD40L, TMZ‐CD40L and 41BBL, or no transgenes. The viruses induced transgene expression post infection before they were killed by oncolysis. Death receptors TRAIL‐R1, TRAIL‐R2 and Fas as well as immunogenic cell death marker calreticulin were upregulated in cell lines post infection. Similarly, caspase 3/7 activity was increased in most cell lines. Interestingly, in CD40 + cell lines there was a significant effect of the TMZ‐CD40L‐encoding viruses indicating activation of the CD40‐mediated apoptosis pathway. Further, these cell lines showed a significant increase of calreticulin, and TRAIL receptor 1 and 2 post infection. However, LOAd viruses induced PD‐L1 upregulation which may hamper anti‐tumour immune responses. In conclusion, LOAd infection increased the immunogenicity of infected tumour cells and this was potentiated by CD40 stimulation. Due to the simultaneous PD‐L1 increase, LOAd viruses may benefit from combination with antibodies blocking PD1/PD‐L1.