Exploring EGFR, Nectin-4, and TROP-2 as Therapeutic Targets for Bladder Cancer Photoimmunotherapy.

BACKGROUND: Non-muscle invasive bladder cancer (NMIBC) has limited therapeutic options and high recurrence rates. Photoimmunotherapy (PIT) enables targeted tumor ablation using antibody-photosensitizer conjugates and light activation. We evaluated EGFR, Nectin-4, and TROP-2 as PIT targets using cysteine-modified antibodies conjugated to the photosensitizer WB692-CB2. METHODS: Antibodies derived from Cetuximab (Cmb, anti-EGFR), Enfortumab (Enf, anti-Nectin-4), and Sacituzumab (Sac, anti-TROP-2) were engineered with T120C and D265C mutations in the heavy chains for site-specific dye conjugation. Binding of the conjugates to BC cells was tested by flow cytometry and light-induced cytotoxicity of the conjugates, alone or in combination, was assessed by viability assays following irradiation. RESULTS: Cysteine-modified antibodies were produced as intact IgG molecules and were efficiently conjugated with WB692-CB2 without loss of antigen specificity. SacT120C/D265C-WB692-CB2 showed the highest target binding and achieved near-complete cell killing at a red-light dose of 32 J/cm2. CmbT120C/D265C-WB692-CB2 required a fourfold higher light dose for comparable efficacy, while EnfT120C/D265C-WB692-CB2 demonstrated lower potency. No cytotoxicity was observed in antigen-negative cells. Combined treatment enhanced cytotoxicity, indicating additive phototherapeutic effects. CONCLUSIONS: Our findings suggest that PIT targeting EGFR, Nectin-4, or TROP-2 merits further preclinical development as a targeted therapeutic approach for NMIBC, including potential combinatorial or personalized strategies.