Chimeric antigen receptor (CAR) T-based immunotherapies have shown great efficacy in treating various cancers and autoimmune diseases. Recent advancement in rapid CAR-T cell therapy has shown that a shortened CAR-T cell manufacturing process reduces cell differentiation and exhaustion. Consequently, this leads to higher potency and anti-tumor activity. This form of rapid cell therapy will significantly help reduce manufacturing costs. Furthermore, it will allow production at near patients’ places (“point-of-care”). This has created a demand for innovative, closed, automated, and space-efficient manufacturing methods.
Here we present a streamlined process that enables the manufacturing of rapid CAR-T cells within 48 hours in a closed system. Starting from fresh/frozen leukopak, we first isolate the cell using GMP CD4/CD8 nanometer magnetic beads on MARS® Bar. This is a novel cell isolation/cell processing platform employing column-free magnetic separation modules with closed fluidics. With the MARS® Bar, we obtain >95% purity of T cells with <0.3% B cell contamination. The system automatically elutes the isolated T cells into complete media at a controlled concentration within a collection bag. Afterward, using a cell incubator, we transfer the T cells collected in the bag to a G-Rex bioreactor or gas-permeable bag for activation and transduction. After 24 hours or 48 hours incubation with CD3/CD28 activation reagent and CAR-19 lentiviral vector, CAR-T cells are ready to harvest. Within 48 hours, CD4/CD8 magnetic beads remained attached to the cells.
Next, the MARS® Bar processes the cells using its magnetic selection in-situ rinse option. This specialized program focuses on rapid cell therapy that enables additional purification of CAR-T cells by removing free viruses. Purified CAR-T cells are eluted directly in harvest saline or cryopreservation solution. This occurs without additional media-exchange. In the harvest step we recovered >90% cells and detected <1% virus residue. CAR-T cells in this process are assessed via killing and potency assays. The results showed comparable outcomes to conventionally manufactured CAR-T cells.
This streamlined process only involves MARS® Bar and a bioreactor- eliminating the use of centrifuge, reducing cost, time, and space requirements. As a result, this process allows high-throughput CAR-T manufacturing at “point-of-care” settings with the benefits of rapid cell therapy.
