Fig. 3
HITI based CRISPR knock-in CAR-T cell manufacturing at clinical scale. a Schematic workflow of leukapheresis processing to manufacture CRISPR KI CAR-T cells at clinical scale. Per Donor 1 × 109 cells were activated and electroporated. Cultures were split up equally and either left untreated or treated with MTX for enrichment. b + c Viability (b) and fold change (c) of respective cultures over time. d Representative quadrant plots (day 14) showing GD2-CAR expression in TRAC positive cells for viral transduced CAR-T cells and TRAC negative cells for GD2 knock-in CAR-T cells. e GD2-CAR frequency over time across all three donors. f expansion of respective GD2-CAR-T cells for different time points normalized to the number of activated T cells. g, Total GD2-CAR-T cell counts for knock-in CAR-T cells (* = Donors with projected numbers after culture split on day 10). h Frequency of viable and dead cells in edited and non-edited T cells after MTX treatment assessed via flow cytometry on day 7. All experiments were conducted with n = 3 independent donors. Error bars indicate SD