A CDK4/6 inhibitor-armed oncolytic adenovirus reverses T cell exhaustion through the Rb-p65-CCL5 pathway and potentiates the antitumor activity of anti-PD-1 or CAR-T therapy in colorectal cancer.

Introduction: The efficacy of oncolytic adenoviruses in colorectal cancer models is constrained by a treatment-induced limitation: the high-dose, repetitive administration required for sustained oncolysis promotes chronic antigen exposure and tumor microenvironmental stress, driving CD8+ T cells into a state of exhaustion. Methods: To mitigate this, we constructed an oncolytic adenovirus, ADV-PTD4-D3, engineered for intratumoral expression of a peptide inhibitor of CDK4/6. This local strategy aims to retain immunomodulatory potential while minimizing systemic exposure. In syngeneic murine models, ADV-PTD4-D3 demonstrated improved tumor control and the ability to induce robust, antigen-specific immunological memory, with its therapeutic effect being primarily dependent on CD8+ T cells. Notably, it also exhibited potent antitumor activity in a humanized xenograft model and showed no evidence of significant off-target toxicity in immunocompetent hosts. Results: The mechanism involves a signaling axis where viral-mediated CDK4/6 inhibition reduces retinoblastoma (Rb) protein phosphorylation. This decrease relieves Rb-mediated sequestration of the NF-kB p65 subunit, allowing p65 nuclear translocation and transcriptional upregulation of the T-cell chemoattractant CCL5, a factor linked to favorable patient prognosis. Thus, ADV-PTD4-D3 promotes a T-cell-inflamed microenvironment by providing a sustained chemotactic signal CCL5 for CD8+ T cell recruitment. Furthermore, this treatment strategy successfully reverses the functional exhaustion of infiltrating CD8+ T cells, thereby addressing two major barriers to effective therapy: inadequate infiltration and functional exhaustion. By modifying the tumor microenvironment in this way, the armed virus addresses two factors that limit T-cell-based immunotherapies: inadequate infiltration and functional exhaustion. Correspondingly, ADV-PTD4-D3 treatment improved the antitumor response to both PD-1 blockade and CAR-T cell therapy in combination studies. Discussion: These findings suggest that engineering oncolytic viruses to locally modulate pathways involved in T cell exhaustion represents a viable and translatable strategy for enhancing antitumor immunity.