Arming oncolytic Coxsackievirus B3 with Neoleukin-2/15 enhances growth inhibition of colorectal carcinomas and induces T cell activation.

Oncolytic viruses (OVs) represent an emerging class of cancer immunotherapeutics. Arming OVs with immunomodulatory transgenes is a promising strategy to reshape the tumor microenvironment (TME) and enhance therapeutic efficacy. We developed the oncolytic Coxsackievirus B3 (CVB3) strain PD-H, which has previously shown antitumor activity in colorectal and pancreatic cancer models. Here, we evaluated the capacity of PD-H to express transgenes and investigated whether its antitumor efficacy in colorectal cancer can be enhanced by insertion of the interleukin-2 (IL-2) mimetic Neoleukin-2/15 (Neo-2/15). Transgene insertion was best tolerated at the VP1-2A junction of the PD-H polyprotein, with viral replication and cytotoxicity inversely correlating with insert size. Genetic stability depended on the host cell line, transgene sequence and transgene length, with inserts up to 350 bp remaining stable for at least 10 viral passages. PD-H-derived Neo-2/15 was biologically active and induced proliferation of human CD4+ and CD8+ T cells in vitro. In vivo, intratumoral administration of PD-Neo-2/15 reduced the growth of subcutaneous Colon-26 tumors more effectively than PD-H and was associated with modulation of the TME, including an increased proportion of CD8+ T cells. Taken together, we demonstrate that arming PD-H with Neo-2/15 is a promising strategy to further enhance its anticancer efficacy.