Cleavage-activatable antibody-photosensitizer conjugate enables tumor-selective photodynamic activation and immunogenic cell death.

Antibody-photosensitizer conjugates (APCs) have emerged as promising platforms for targeted photodynamic therapy (PDT); however, most conventional APCs employ non-cleavable linkers and remain constitutively photoactive, generating fluorescence and phototoxicity irrespective of their biological location. This "always-on" behavior compromises imaging specificity and increases the risk of off-target phototoxicity. Here, we report an enzyme-responsive, cleavage-activatable cetuximab-photosensitizer conjugate (Q-Cet) that enables tumor-selective photodynamic activation and tumor-restricted immunogenic cell death (ICD) in epidermal growth factor receptor (EGFR)-overexpressing lung cancer. Q-Cet incorporates a cathepsin B-cleavable linker integrating chlorin e4 (Ce4), a tryptophan-mediated photoinduced electron transfer (PET) quencher, a protease-sensitive peptide substrate, and a zwitterionic antifouling moiety, allowing molecular "off-on" control of fluorescence and photodynamic activity. Following EGFR-mediated internalization, lysosomal cathepsin B cleavage disrupts PET quenching, thereby restoring fluorescence and singlet oxygen generation selectively within tumor cells. Importantly, this activation-dependent photodynamic response induces robust ICD exclusively in cancer cells, as evidenced by extracellular ATP release, calreticulin (CRT) exposure, and high mobility group box 1 (HMGB1) translocation, thereby promoting spatially confined antitumor immune activation. In vivo, Q-Cet-mediated PDT achieved significant tumor growth suppression upon light irradiation, with minimal off-target toxicity and favorable biocompatibility. Collectively, this study establishes a cleavage-activatable APC strategy that enables precise molecular control of photodynamic activity and tumor-restricted immune activation, providing a promising approach for enhancing the safety and specificity of antibody-based phototherapy in EGFR-overexpressing malignancies.