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

Background: Non-muscle invasive bladder cancer (NMIBC) has limited therapeutic op-tions and high recurrence rates. Photoimmunotherapy (PIT) enables targeted tumor abla-tion 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, an-ti-EGFR), Enfortumab (Enf, anti-Nectin-4), and Sacituzumab (Sac, anti-TROP-2) were en-gineered with T120C and D265C mutations in the heavy chains for site-specific dye con-jugation. Binding and light-induced cytotoxicity of the conjugates, alone or in combina-tion, were assessed in BC cells by flow cytometry and viability assays following irradia-tion. Results: Cysteine-modified antibodies were produced as intact IgG molecules and ef-ficiently 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 showed lower potency. No cytotoxicity was observed in antigen-negative cells. Combined treatment en-hanced cytotoxicity, indicating additive phototherapeutic effects. Conclusions: SacT120C/D265C-WB692-CB2 represents a highly effective PIT conjugate for NMIBC. Our find-ings support PIT as a promising, targeted, and minimally invasive therapeutic option for BC, potentially adaptable for combinatorial or personalized strategies.