Fig. 3

Nanoparticles can knockout T cell receptors in CAR-programmed lymphocytes. a Integration of nanoparticle (NP) transfection into normal manufacturing of CAR-T-cells. After stimulation with anti-CD3/CD28-coated beads (day 0), CD8-targeted mRNA NPs were introduced on days 1 and 2, then lentiviral transduction with a vector encoding the leukemia-specific 19-41BBz CAR was performed on day 3. We added either NPs carrying mRNAs encoding megaTAL nuclease plus eGFP, or control particles loaded with eGFP mRNA alone. b Flow cytometry of NP transfection efficiencies (based on eGFP signals) correlated with surface expression levels of TCRs (based on CD3 signals) by T-cells following NP treatments. c Summary plot showing editing efficiency as measured by loss of CD3 surface expression at day 14 (n = 6). d Surveyor assay confirming TCRα chain gene locus disruption. e Flow cytometry of lentiviral transduction in genome-edited versus control T-cells. f Bar graph showing mean viral transductions and SE of three independent experiments conducted in duplicate; n.s., not significant g, h Proliferation and cytolytic activity of TCR+ (FACS sorted TCR-positive, unedited 19-41BBz CAR-T-cells) and TCR- (FACS sorted TCR-negative, genome edited) 19-41BBz CAR-T-cells. To measure proliferation, T-cells were co-cultured on irradiated TM-LCL leukemia cells. Cytolytic assays were performed with CD19-expressing K562 target cells. i T cell IFN-γ release was measured with ELISA 48 h after stimulation on CD19+ TM-LCL leukemia cells or control LNCaP C4-2 prostate adenocarcinoma cells. Data from two experiments run in triplicate are shown