Safety profile associated with the SB system.

(A) Telomere length of cells was measured using fluorescence in situ hybridization and flow cytometry (Flow-FISH) assay. Predominant T cell population at day 28 (V1 and V2, CD8⁺ T cells; V3, CD4⁺ T cells) was compared to respective miltenyi column purified subset of T cells from day 0. Mean ± SD of triplicates for each validation run is represented. (B) Genomic DNA from CAR⁺ T cells at day 28 was amplified using primers and probes specific for CD19RCD28 CAR. Relative Quantity (RQ) analyses of the CD19RCD28 target copy number was determined using normal donor PBMC as reference and endogenous RNaseP as a normalizer. Mean ± SD of triplicates for each validation run is shown. (C) TCR Vβ analysis of day 28 and day 35 CAR⁺ T cells. Data shows mean ± SD of three validation run CAR⁺ T cells as compared to day 0 unmanipulated controls. (D) A representative genomic PCR showing lack of SB11 transposase integration. Genomic DNA (20 ng) was amplified using SB11 or GAPDH primers. CAR^neg control T cells (lane 5) and CAR⁺ T cells (lane 7) amplified using SB11 primers; CAR^neg control T cells (lane 6), CAR⁺ T cells (lane 8) and Jurkat stably expressing SB11 (lane 4) amplified using GAPDH primers. Jurkat stably expressing SB11 (Jurkat/SB11-IRES2-EGFP) (lane 3) and the linearized plasmid, pKan-CMV-SB11 (lane 2) amplified using SB11 primers were used as positive controls. (E) G-banded karyotypes of CAR⁺ T cells from the three validation runs reveal no structural or numeric alteration. A representative spread from validation 2 is shown.