Light-activated EGFR antibody–drug conjugate: Analysis of selectivity and translational profile for first-in-human evaluation.

3023 Background: Conventional antibody–drug conjugates (ADCs) are limited by systemic toxicity and narrow therapeutic windows. Cetuximab-I21-2 is a first-in-class, EGFR-targeted ADC conjugated to a Heloporfin-derived photosensitizer engineered for activation exclusively at the tumor site using red-light irradiation. This approach enables biologically targeted delivery with externally controlled cytotoxic activation, designed to improve the therapeutic index and support safe translation into first-in-human studies. Methods: In vitro cytotoxicity was evaluated across EGFR-high, intermediate, and low tumor cell lines (A431, HCC827, Huh7, NCI-N87, MIA-PaCa-2) with or without red-light activation (1–5 J/cm²). Cellular ROS generation, mitochondrial membrane potential disruption, and photodynamic injury were assessed by live-cell imaging. In vivo activity was tested in A431 xenograft-bearing B-NDG mice following a single intravenous dose of Cetuximab-I21-2 (35.3 mg/kg) and localized tumor irradiation at 100 or 200 J. Safety evaluation included body-weight trends and clinical observations. Results: Cetuximab-I21-2 showed negligible dark toxicity up to several µg/mL, confirming that the payload remains inactive without illumination. Upon irradiation, the ADC demonstrated potent, energy-dependent cytotoxicity (IC50: 0.05 µg/mL in A431 and 0.14 µg/mL in HCC827 at 5 J/cm²), with reduced effects in EGFR-low models, supporting a target- and activation-dependent mechanism. Illumination triggered rapid singlet-oxygen and ROS production and immediate mitochondrial depolarization. In vivo, Cetuximab-I21-2 achieved irradiation-dependent tumor suppression, reaching 22.2% inhibition at 200 J, and produced histologic photodynamic injury localized to the tumor. Treatment was well tolerated: animals exhibited only transient (<10%) weight loss and no systemic toxicity. Conclusions: Cetuximab-I21-2 demonstrates a strong translational profile with selective activation, robust preclinical antitumor activity, and a favorable safety margin. The spatial control offered by light activation may enable dose-sparing strategies and reduce systemic adverse events typically associated with ADCs. These findings support advancement toward IND-enabling studies, including optimization of clinical illumination parameters, device–drug integration, and early feasibility planning for first-in-human evaluation in EGFR-expressing solid tumors.