Fig. 3.

In vitro cytotoxicity of αPSMA/αCD3 conjugates in the presence of freshly purified unactivated hPBMCs. (A) Conjugates show dose-dependent activity on PSMA⁺ C4-2 cells with different EC₅₀ values, whereas the unconjugated mixture shows no activity. (B) Conjugates have no effect on PSMA⁻ DU-145 cells. Cytotoxicity was detected by LDH-release assay. (C) Proinflammatory cytokine TNF-α levels in the supernatant of the cytotoxicity assay. Dose-dependent TNF-α signals were detected by ELISA only in the presence of PSMA⁺ C4-2 cells. RFU, relative fluorescence units. (D) Significantly reduced cytotoxicity of C4-2 cells was observed in the competition assay in the presence of either the competitive antagonist 2-phosphonomethyl pentanedioic acid (2-PMPA) or αCD3 Fab. No cytotoxicity was observed in the absence of hPBMCs. RAU, relative absorbance units.