Fig. 3

A ddPCR sensitivity assessment by plasmid dilutions. Both plasmids were diluted in nontransduced CEM cell gDNA, and CEM-CAR was diluted in an untransduced CEM cell line. ddPCR (28z and 28BBz) was applied to plasmid dilution (top) and to gDNA extracted from the cell line dilution (bottom). Positive and negative droplets were delimited by the threshold (pink line). The CAR copy number is plotted against plasmid copy numbers or cell dilution factors. Linear regression curves and correlation coefficients are provided for n = 4 independent experiments. CAR DNA plasmids (10e6 copies) or CAR + cell lines (undiluted) are considered as positive PCR controls. NT: not transduced sample. B Vector copy number. (vector copy number (VCN, transgene copies/CAR T cells, Y left axis, red circles) is plotted against multiplicity of infection (MOI) calculated from 28z and 28BBz ddPCR and reference housekeeping gene (GAPDH) ddPCR normalized to the transduction efficiency percentage. Transduction efficiency (TE, blue squares) obtained by flow cytometry (FC, %) is provided (Y right axis). (Left) Average VCN measured for axi-cel (n = 4) and tisa-cel (n = 8) obtained from leftover bags of CAR T cell products and for IL-1RAP CAR T cells produced under experimental (n = 4) or GMP-like conditions (n = 4). *p < 0.05