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

The adenovirus <i>E3</i> region's immune-modulating genes (<i>gp19K</i>, <i>Adenovirus Death Protein [ADP]</i>, <i>E3B</i>) are frequently modified in oncolytic adenoviruses (OAds) through deletion and transgene insertion like granulocyte-macrophage colony-stimulating factor (<i>GM-CSF</i>). 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 (del<i>gp19K</i>), M20 (del<i>gp19K</i> and <i>ADP</i>), M22-0 (del<i>gp19K</i> and <i>E3B</i>), and their <i>GM-CSF</i>-armed derivatives, systematically evaluating the impact of <i>ADP</i> and <i>E3B</i> deletions on viral replication, tumor cell lysis, immune modulation, and <i>in vivo</i> antitumor activity. Key findings revealed that <i>gp19K/ADP</i> 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. <i>Gp19K/E3B</i> 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, <i>gp19K/ADP</i> 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 <i>gp19K/ADP</i> deletion in optimizing viral replication, transgene expression, and immune evasion, providing a strategic framework for engineering next-generation OAds.