Abstract 5638: A first-in-class photo-responsive EGFR-ADC drives ROS-mediated, energy-dependent tumor eradication in preclinical models

Abstract Light-activated antibody-drug conjugates (ADCs) represent an emerging class of precision therapeutics that enable spatial and temporal control of cytotoxicity. By restricting payload activation to illuminated tumor sites, this modality can enhance local potency while limiting systemic toxicity. We developed Cetuximab-I21-2, a next-generation EGFR-targeted ADC incorporating a Heloporfin-derived photosensitizer engineered for high singlet-oxygen yield, serum stability, and rapid photochemical activation upon exposure to red light near 650 nm. This architecture integrates receptor-mediated tumor localization with externally programmable activation, enabling precise, on-demand cytotoxicity.In vitro studies were conducted across tumor cell lines with high, intermediate, and low EGFR expression, including A431, HCC827, Huh7, NCI-N87, and MIA-PaCa-2. Cetuximab-I21-2 exhibited minimal dark toxicity at concentrations up to several micrograms per milliliter, confirming that the payload remains inactive without illumination. Upon irradiation, the ADC produced potent, energy-dependent cytotoxicity consistent with singlet-oxygen-driven membrane injury and mitochondrial dysfunction. IC50 values were 0.05 µg/mL in A431 and 0.14 µg/mL in HCC827 at 5 J/cm2, with substantially reduced potency in EGFR-low cells, demonstrating both antigen-dependent delivery and illumination-triggered activation. Fluorescent ROS probes showed an immediate burst of intracellular singlet oxygen and secondary reactive oxygen species within minutes of irradiation, accompanied by rapid loss of mitochondrial membrane potential and plasma membrane integrity, confirming the dual targeting-activation mechanism.In vivo antitumor activity was evaluated in A431 xenograft-bearing B-NDG mice treated with a single 35.3 mg/kg intravenous dose followed by localized irradiation at 100 or 200 J. Cetuximab-I21-2 produced distinct, energy-dependent tumor suppression, achieving 22.2% inhibition at 200 J. Histopathology revealed focal necrosis and photodynamic vascular disruption, consistent with ROS-mediated tissue injury. Treated animals maintained stable clinical condition, and transient (<10%) weight loss resolved spontaneously without supportive care, with no evidence of systemic toxicity.Collectively, these findings demonstrate that Cetuximab-I21-2 provides potent, selective tumor ablation through the integration of EGFR-targeted delivery, payload stability, and externally controlled photochemical activation. This first-in-class photo-responsive ADC platform offers a programmable therapeutic strategy that may improve therapeutic index, enable dose-sparing treatment, and expand precision oncology approaches for EGFR-expressing solid tumors. Citation Format: Wentao Shang, Hua Shang, Josie Cai, Vicky Qin, Lyan Chen, Xiaobo Zhang. A first-in-class photo-responsive EGFR-ADC drives ROS-mediated, energy-dependent tumor eradication in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5638.