Targeted E3 Region Engineering Boosts Antitumor Efficacy of Conditionally Replicating Adenoviruses in an Immunocompetent Tumor Model.

The adenovirus E3 region's immune-modulating genes (gp19K, Adenovirus Death Protein [ADP], E3B) are frequently modified in oncolytic adenoviruses (OAds) through deletion and transgene insertion like granulocyte-macrophage colony-stimulating factor (GM-CSF). However, the synergistic effects of dual-gene deletions on antitumor efficacy and transgene capacity remain unexplored. To address this, we constructed three E3-modified OAds including OAd5-delgp19K (delgp19K), M20 (delgp19K and ADP), M22-0 (delgp19K and E3B), and their GM-CSF-armed derivatives, systematically evaluating the impact of ADP and E3B deletions on viral replication, tumor cell lysis, immune modulation, and in vivo antitumor activity. Key findings revealed that gp19K/ADP deletion OAd prolonged intracellular viral replication, creating a "viral bomb" effect that delayed cell lysis, evading anti-adenovirus antibodies, sustained GM-CSF expression, and culminating in superior tumor suppression. Gp19K/E3B deletion OAd accelerated viral dissemination but triggered rapid antibody-mediated clearance in immunocompetent hosts, resulting in transient GM-CSF expression and diminished therapeutic persistence. In immunocompetent Syrian hamster Hap-T1 subcutaneous tumor models, gp19K/ADP deletion OAd demonstrated potent tumor inhibition, durable immune microenvironment remodeling, robust viral replication, and evading anti-adenovirus antibodies. These results underscore the critical role of coordinated gp19K/ADP deletion in optimizing viral replication, transgene expression, and immune evasion, providing a strategic framework for engineering next-generation OAds.