CAR T cell manufacturing

For in vitro assays, buffy coats from healthy donors were obtained from DRK Dortmund. Density gradient centrifugation with Pancoll human (PAN-Biotech, catalog no.: P04-601000) was used for PBMC isolation. T cells were isolated from PBMCs with the Pan T Cell Isolation Kit, human (Miltenyi Biotec, catalog no.: 130-096-535). Isolated T cells were cultured in TexMACS™ Medium (Miltenyi Biotec, catalog no.: 130-097-196) supplemented with 12.5 ng/mL IL-7 (Miltenyi Biotec, catalog no.: 170-076-111) and 12.5 ng/mL IL-15 (Miltenyi Biotec, catalog no.: 170-076-114). For activation before transduction, T cells were seeded in a 24-well plate at a density of 1 × 10⁶ cells/mL and T Cell TransAct™ (Miltenyi Biotec, catalog no.: 130-111-160) was added. After 24 h, the cells were transduced with an MOI of 10. After 3 d, the culture medium was exchanged completely to remove residual LVV particles. T cells were cultured for 10–14 d with the regular addition of fresh culture medium every 2 d. CAR T cells for in vivo studies were manufactured using the CliniMACS Prodigy™ (Miltenyi Biotec) as described previously by Lock et al.³⁰ As starting material leukapheresis obtained from “Institut für klinische Transfusionsmedizin und Immungenetik Ulm” was used. The enriched T cells were transduced at an MOI of 10 and cultured until day 10 for harvesting.