A PRK-armed oncolytic adenovirus drives calreticulin exposure for dendritic cell licensing to prime antitumor CD8⁺ T cells and synergizes with anti-PD-1 or CAR-T therapy in colorectal cancer.

BACKGROUND: The therapeutic potential of oncolytic adenoviruses (ADVs) in colorectal cancer is constrained by a counterproductive host response: virus-triggered hyperactivation of the pro-survival AKT pathway, which fosters an immunosuppressive tumor microenvironment. METHODS: We engineered a novel oncolytic adenovirus, ADV-PRK, designed to co-express the AKT-inhibitory peptide PRK. Its antitumor efficacy and mechanisms were evaluated in syngeneic (MC38, CT26) and humanized mouse models. Molecular analyses (Western blot, immunofluorescence, and qPCR) elucidated signaling pathways. Immunological outcomes were assessed via flow cytometry and in vitro co-culture assays. Combination therapies with anti-PD-1 or CAR-T cells were tested in vivo. RESULTS: We demonstrate that ADV-PRK abrogates phosphorylation of both AKT and its downstream effector, the ER calcium channel IP3R3. This suppression drives ER calcium efflux, inducing calreticulin (CRT) translocation to the cell surface. Surface-exposed CRT acts as a potent "eat-me" signal, licensing dendritic cells to prime and activate tumor-specific CD8⁺ T cells, which are essential for efficacy. In vivo, ADV-PRK monotherapy achieved superior tumor control. It demonstrated potent synergy with both anti-PD-1 and CAR-T cell therapy, by remodeling the immune landscape to expand activatable T cells and support adoptive cellular therapy. Its activity in a humanized model confirms translational relevance. CONCLUSIONS: Our work delineates a distinct immunomodulatory axis, AKT-IP3R3-Ca2⁺-CRT, to reverse adenovirus-induced immunosuppression. This provides a translatable, mechanism-based strategy to convert immunologically "cold" tumors into environments receptive to immunotherapy, thereby offering a rational combinatorial approach to overcome resistance and significantly broaden the applicability of current immunotherapies.