The recent evolution of oncolytic virotherapy has yielded viral platforms with enhanced tumor tropism and expanded engineering flexibility, thereby enabling not only direct oncolysis but also deliberate promotion of antitumor immune responses. Here, we systematically explore multiple insertion sites for calreticulin (CALR) within an oncolytic adenovirus, identifying the most optimal variant that exposes robust CALR on tumor cell membrane and functionally motivates macrophages in addition to directly mediating tumor cell lysis. Mechanistically, this variant incorporates a precise deletion within the E1A CR2 domain (920–946 bp), enabling selective replication in retinoblastoma-deficient tumor cells. Furthermore, the E3-gp19k region has been replaced with an exogenous Cytomegalovirus (CMV) promoter to achieve precise regulation of CALR overexpression and diminish systemic toxicity. Functionally, by bulk-RNA-Seq, we demonstrate that oncolytic adenovirus (oAd)-CMV-CALR transfection induces endoplasmic reticulum stress, as evidenced by upregulation of phosphorylated eIF2α, which facilitates the translocation of CALR to the plasma membrane. Via ex vivo coculture assays, we validate that oAd-CMV-CALR transfection enhances the phagocytic capacity of M2 macrophages and promotes their repolarization toward an M1-like phenotype. These findings are further validated in patient-derived ovarian cancer spheroids, underscoring the translational potential of our approach. In vivo, oAd-CMV-CALR suppresses Hepa1-6 xenograft growth, boosts CD8+ T-cell infiltration, and exhibits favorable safety. Collectively, our findings highlight oAd-CMV-CALR as a potential therapeutic approach to modulate the tumor microenvironment and improve cancer immunotherapy outcomes.
