Chimeric antigen receptor (CAR) T-based immunotherapies have shown great efficacy in treating various cancers as well as autoimmune diseases. Recent advancement has shown that shortened CAR-T cell manufacturing process reduces cell differentiation and exhaustion, leading to higher potency and anti-tumor activity. This process will contribute greatly to lowering the manufacturing cost and production at near patients’ places (“point-of-care”, thus has created a demand for innovative, closed, automated, and space-efficient manufacturing method.
Here we present a streamlined process which enables the manufacturing of CAR- T cells within 48 hours in closed systems. Starting from fresh/frozen leukopak, we first did immunomagnetic cell isolation using GMP CD4/CD8 nanometer magnetic beads on MARS® Bar, a novel cell isolation/cell processing platform employing column-free magnetic separation modules with closed fluidics. We obtained >95% purity of T cells with <0.3% B cell contamination. The isolated T cells were eluted with complete media automatically in the controlled concentration in a bag. T cells collected in the bag were transferred to G-Rex bioreactor or gas-permeable bag for activation and transduction in a cell incubator. After 24 hours or 48 hours incubation with CD3/CD28 activation reagent and CAR-19 lentiviral vector, CAR-T cells were ready to harvest. As within 48 hours CD4/CD8 magnetic beads were still attached to cells, we ran cells though MARS® Bar with magnetic selection in-situ rinse option, which is the special program allowing extra purification of the CAR-T cells from free viruses. Purified CAR-T cells were eluted in harvest saline or cryopreservation solution directly without additional media-exchange. In the harvest step we recovered >90% cells and had detected <1% virus residue. CAR-T cells produced from this process were assessed via killing and potency assays, showing comparable results to conventionally manufactured CAR-T cells.
This streamlined process only involves MARS® Bar and a bioreactor, eliminating the use of centrifuge, thus reducing costs, time, and space requirements. This process allows high-throughput CAR-T manufacturing at “point-of-care” settings.
