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Chimeric antigen receptor (CAR) T-cell therapy has shown limited efficacy against solid tumors due to antigen heterogeneity and scarcity of tumor-specific targets.

To address those challenges, we report a covalent CAR-T strategy that achieves programmable tumor recognition via oncolytic adenovirus-mediated (OAD) delivery of artificial antigens. An engineered OAD was designed to induce tumor-selective expression of a membrane-anchored SpyTag-containing artificial antigen on infected tumor cells. In parallel, we generated SpyCatcher CAR-T cells by replacing the conventional single-chain variable fragment (scFv) with SpyCatcher, which forms a spontaneous covalent bond with SpyTag and redirects CAR-T-cell activity toward virus-labeled tumor cells.

In vitro, optimized SpyCatcher CAR-T cells mediated selective cytotoxicity against SpyTag-positive tumor cells, achieving >85% specific lysis at an effector-to-target ratio of 1:1, while sparing antigen-negative cells. The OAD efficiently induced tumor-selective expression of membrane-anchored SpyTag-fused antigens across multiple cell lines. Combined treatment with OAD and SpyCatcher CAR-T cells resulted in substantially greater antitumor activity than either monotherapy alone. In vivo, the combinatorial strategy significantly inhibited tumor growth and increased intratumor CD3⁺, CD8⁺ T-cell infiltration in both immunodeficient and immunocompetent mouse models. Importantly, patient-derived prostate cancer organoids were effectively transduced by OAD and supported robust SpyCatcher CAR-T cell infiltration and cytotoxicity, demonstrating the translational potential of this approach.

This study establishes a modular platform for solid tumor immunotherapy therapy by integrating covalent SpyCatcher CAR-T cells with SpyTag-delivering OAD. By decoupling tumor recognition from endogenous antigen expression, this approach provides a generalizable strategy to overcome antigen heterogeneity and scarcity of tumor-specific targets in solid tumors.