Anti-PSMA CAR-T Cell Generation

The anti-PSMA CAR/eGFP lentiviral transfer vector (LV) and viral particle production in 293 T cells have been previously described (19 (link)). We used PBMC from healthy donors to generate CAR-T cells. PBMC were activated for 48 hours (hrs) with OKT-3 (50 ng/mL; Ortho Biotech Inc) and human IL-2 (hIL-2, 300 U/mL; Proleukin; Novartis Pharmaceuticals). Then, T cells were transduced by LVs with a TU/ml infection of 05-5 x10⁷, as previously described (19 (link)). Briefly, the viral supernatant was added to T cells for 18 hours at 37°C and 5% CO₂, with protamine sulfate (40 mg/mL; Sigma- Aldrich) and hIL-2 (500 U/mL). Fresh complete medium containing hIL-2 (100 U/mL) was then replaced to the viral supernatant. Seventy-two hrs later, we analyzed CAR and eGFP expression. Every week CAR-T cells were stimulated with irradiated (60 Gy) PC3-PSMA at a 10:1 ratio. Complete medium with fresh IL-2 was changed every 3 days.