Generation and Transduction of Anti-Mesothelin CAR T Cells

The anti-mesothelin M5 4-1BB-zeta CAR was previously described (25 (link), 29 (link), 30 ). CD4 and CD8 T cells were combined at a 1:1 ratio and rested overnight with R10 media supplemented with 5ng/mL human IL7 and 5ng/mL human IL15 (Preprotech). The next day, CRISPR-Cas9 editing was performed, and cells were cultured in cytokine-supplemented R10 media at 5 × 10⁶ cells/mL at 37 °C and activated 4 to 6 h later with Dynabeads® CD3/CD28 CTS™ (Thermofisher) at a 3:1 bead-to-cell ratio at 1 × 10⁶ cells/mL. After 24 h, T cells were transduced at a multiplicity of infection (MOI) of 3. At day 5, beads were removed from cultures. T cell cultures were maintained at 6 × 10⁵ cells/mL. Cell number and volume were monitored daily using Multisizer 3 Coulter Counter (Beckman). Transduced T cells were cryopreserved when they reached a rested state, as determined by cell volume.