Fig. 1.

Regulated PD-1 blockade in ROR1 CAR-T cells—an inducible system. A Transgene schematic of ROR1-targeted F CAR lentiviral construct. B F CAR-mediated killing was assessed at 72 h at 10:1 target/effector (T/E) ratio, mean + SD of 3 donor-derived CAR-T cells. C PD-1 expression of F CAR-T cells assessed via flow cytometry at 0 h (without target cells) and every 24 h during co-culture with the cell lines MDA-MB-231 and H1975 (both ROR1 +) or the ROR1-negative cell line SUPT1 at a 10:1 T/E ratio. Results show mean + SD of 3 donor-derived CAR-T cells. D Transgene schematic of the F i-CAR lentiviral construct. E Supernatant from co-cultures was collected and secreted anti-PD-1 scFv quantified via PD-1 ELISA. Results show mean ± SD of 3 donors in triplicates. F Flow cytometry binding assay of secreted aPD-1 scFv to SupT1-PD-1 + cells shows specific binding represented by PE-anti-His Tag shift (right plot, blue histogram) compared to background, negative control (supernatant from F CAR co-culture, light blue histogram) and non-induced secretion (F i-CAR with ROR1 cell co-culture, orange histogram). G Long-term 72 h co-cultures of F CAR-T cells, F i-CAR-T cells or F CAR-T cells CAR + 10 µg/mL anti-human PD-1 antibody (F CAR + mAb), against MDA-MB-231 and H1975 cell lines, mean ± SD of 3 donors normalized to negative control CD19 CAR; two-way ANOVA used: *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001