AUTOMATION OF RAPID CAR T CELL MANUFACTURING ON A SINGLE PLATFORM

Background & Aim Autologous cell therapy using genetically modified T cells to express chimeric antigen receptor (CAR) has yielded durable responses in patients. However, currently only a small portion of patients are privileged to receive the available therapies. High cost and long manufacturing time contribute to the key limitations on a broader adoption. Researchers have recently demonstrated a process of generating functional CAR T cells within 24 hrs with greater potency. This rapid process provides a new hope in making CAR T therapy affordable and accessible. The reported rapid manufacturing process is still manual operation and involves use of multiple tools. To automate the rapid process for quality cell production, we developed a rapid CAR T manufacturing process on a single platform. Methods, Results & Conclusion Rapid process eliminates in vitro cell expansion and requires much smaller doses compared to conventional process. Manufacturing of CAR-T cells is simplified to: (1) T cell selection, (2) viral transduction, and (3) cell purification. Fresh peripheral blood can now replace leukopak as starting material. We conducted feasibility study on a platform equipped with 3x magnetic cell separation modules. Magnetic cell separation process is column-free automated operation within enclosed sterile lines. First, T cells were isolated directly from peripheral blood from a healthy donor using CD4/CD8 nanomagnetic beads. Then isolated T cells were released using cell culture medium. eGFP expressing lentivirus were added to the enriched T cells with other reagents required for viral transduction. After 20 hrs incubation, transfected T cells were purified on a second magnetic module to remove free viruses and transduction medium. T cells were then released and collected in saline solution ready for injection. Starting from 20mL of peripheral blood, we isolated T cells with 95% purity and 70% recovery. >99% of lentivirus was rinsed off during purification. After transduction, >17.5% T cells became GFP positive, with 80% viable cells. Compared to reported process, single platform process saves multiple cell washing steps, achieves cell recovery and purity, and reasonable transduction efficiency. In summary, we demonstrated rapid manufacturing of CART cells on an automated platform is feasible. The platform can be the solution for rapid autologous manufacturing of CAR T cells at close-to-patient settings. Autologous cell therapy using genetically modified T cells to express chimeric antigen receptor (CAR) has yielded durable responses in patients. However, currently only a small portion of patients are privileged to receive the available therapies. High cost and long manufacturing time contribute to the key limitations on a broader adoption. Researchers have recently demonstrated a process of generating functional CAR T cells within 24 hrs with greater potency. This rapid process provides a new hope in making CAR T therapy affordable and accessible. The reported rapid manufacturing process is still manual operation and involves use of multiple tools. To automate the rapid process for quality cell production, we developed a rapid CAR T manufacturing process on a single platform. Rapid process eliminates in vitro cell expansion and requires much smaller doses compared to conventional process. Manufacturing of CAR-T cells is simplified to: (1) T cell selection, (2) viral transduction, and (3) cell purification. Fresh peripheral blood can now replace leukopak as starting material. We conducted feasibility study on a platform equipped with 3x magnetic cell separation modules. Magnetic cell separation process is column-free automated operation within enclosed sterile lines. First, T cells were isolated directly from peripheral blood from a healthy donor using CD4/CD8 nanomagnetic beads. Then isolated T cells were released using cell culture medium. eGFP expressing lentivirus were added to the enriched T cells with other reagents required for viral transduction. After 20 hrs incubation, transfected T cells were purified on a second magnetic module to remove free viruses and transduction medium. T cells were then released and collected in saline solution ready for injection.