Prodigy device

Lentiviral supernatant was produced after triple transfection of the HEK293T cell line. Experimental CAR T cells were produced from healthy donors as previously described [11 (link)]. Briefly, cells were transduced after an initial activation and CD3+ MACS-selection step using CD3/CD28 beads following an expansion period in the presence of IL-2. Preclinical-grade IL-1RAP CAR T cells were also produced from healthy donors in a Prodigy device (Miltenyi Biotech) according to the manufacturer’s protocol. Briefly, selected CD4+ and CD8+ cells were activated with CD3/CD28+ IL-7/IL-15 (MACS GMP T Cell TransAct, Miltenyi Biotech) before being transduced and expanded for 9 days in a closed tubing set and TexMACS medium (Miltenyi Biotech). In both cases, genetically modified T cells were purified to increase purity to > 99%.

CAR T cell production was performed on the Prodigy device (Miltenyi). Pheresis was washed in DPBS, stimulated with transact (Miltenyi) and cultured in IL-7 and IL-15. Transduction was performed on d 3 after stimulation initially in retronectin-coated bags, but subsequently was performed in suspension within the Miltenyi Prodigy bioreactor facilitated by vectofusin (Miltenyi). T cells were expanded in the Prodigy device until the dose was reached. Total production time ranged from 7 to 14 d. Products were cryopreserved in DMSO containing cryoprotectant.