Fig. 2

Optimization of Methotrexate (MTX) based selection of CRISPR knock-in GD2-CAR-DHFR-FS T cells. a GD2-CAR-DHFR-FS nanoplasmid design incorporating a gRNA cut site for linearization of the nanoplasmid and dsDNA break in TRAC with correct transgene insertion indicated. b Experimental layout for optimization of MTX enrichment. MTX treatment from day 7–14 has previously been reported to result in efficient enrichment in viral transduced CAR-DHFR-FS T cells and served as a reference. c Titration of MTX in primary human T cells with efficient killing starting at 50 nM MTX (n = 2 independent donors analyzed in technical duplicates). d Comparison of knock-in frequency determined via flow cytometry on day 14 in GD2-CAR-DHFR-FS T cells either non-enriched, enriched from day 3–10 or from day 7–14 (n = 5 independent donors). e MTX time course after CRISPR knock-in starting on day 3 for up to 7 days with plateaued enrichment after 4 days of treatment. All samples were assessed via flow cytometry on day 14 (n = 2 independent donors). f Quadrant plots indicating TCR-a/b and GD2-CAR surface expression for two representative out of five independent donors non-enriched, enriched from day 3–7 and from day 7–14. Flow cytometry was conducted on day 14. g GD2-CAR-T cell yield at day 14. Fold changes were calculated based on number of electroporated T cells on day 2 (n = 5 independent donors). Experiments in d and g were evaluated for statistical significance by paired, two-tailed t tests. Error bars indicate SD