| Vector | lentivirus |
| Transgene/Inserted gene | a scFv from a fully-human anti-CD19 monoclonal antibody; CD8α hinge; transmembrane domains; a CD28 costimulatory domain; a CD3ζ activation domai |
| Vector production method | On day 0, fresh or thawed PBMC were placed in complete medium containing AIM-V CTS™ medium (Thermo/Life Technologies), 5% heat-inactivated pooled human AB serum (Valley Biomedical, Winchester VA), 300 IU/mL interleukin-2 (IL-2) (Proleukin; Prometheus Laboratories), and 50 ng/mL anti-CD3 (MACS® GMP CD3 pure, clone OKT3, Miltenyi Biotech). Cells were incubated in cell culture bags (Origen Biomedical) for 22-24 hours in a 37⁰C, 5% CO2 humidified incubator. On day 1, LSIN-Hu19-CD828Z vector was thawed and added, along with protamine sulfate, to the cell culture bags containing activated T cells. Cells were incubated 44-48 hours in a 37⁰C, 5% CO2 humidified incubator. After the incubation, on day 3 of culture, cells were removed from cell culture bags and resuspended in fresh medium at 0.5-1×106 cells/mL. On day 5, cells were counted and diluted to 0.5-1×106 cells/mL. Cultures were continued until day 7 to day 9 when cells were harvested, concentrated, washed either manually or on a COBE 2991 cell processor, and cryopreserved. The method of washing and concentration was determined by the number of cells in the cultures. CAR T cells were dosed as a specific number of CAR+CD3+ cells/kg of patient bodyweight. |
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