Development of a closed system process for purifying naive CD8+ cells, culturing and transducing with a CD19/22 chimeric antigen receptor (CAR) to produce a clinical T memory stem cell product directed against B cell malignancies

Background & Aim Currently in the Center for Cellular Engineering at the National Institutes of Health, a clinical trial which involves purifying CD8+CD62L+CD45RA+ naive T cells, culturing and transducing with a retroviral CD19 CAR, is underway. Mononuclear cells are enriched from apheresis product by automated density gradient, followed by 3 separate Strep-tactin® magnetic microbead manual selections. The selected Naive T cells are then transduced and cultured for 7 days in gas permeable cell culture bags. The entire process is open, especially purification procedures which are extremely labor intensive, takes between 16-18 hours and is performed over two days. Therefore, a less labor intensive, more streamlined and closed system process is needed. Methods, Results & Conclusion We developed a more efficient and GMP compliant process for generating CD19 and CD22 CAR Tscm product. A large scale CD8+ selection was performed on the CliniMacs Prodigy® platform followed by sterile, closed system cell sorting on the MACSQuant Tyto® for the purification of naive T cells (targets). These purified cells were placed back on the the Prodigy for culture and transduction using an automated customized program. While these enrichments worked well for donors with relatively normal target percentages, several older donors with low targets did not reach the desired purity. We then adopted a CD8+ releasable microbead selection, performed on the CliniMacs Plus device, followed by a CD62L+ microbead selection (Miltenyi Biotec) on the CliniMacs Prodigy to further enrich for target cells. We have performed many small scale selections and sorts and can consistently achieve 80-90% purities utilizing two selections. New MACSQuant Tyto® HS cartridges are now available which could potentially cut the sorting time in half, and along with new recently developed software program to decrease user setting manipulations, resulting in decreased sorting times and increased purities. In addition, after optimizing culture conditions in the CliniMacs Prodigy, as few as 25 million naive T cells can be initiated for culture, and a naive phenotype can be retained after 7 days in culture with a single lentiviral transduction. In summary, we developed a robust GMP compliant process for Tscm purification, culture and transduction which can be used to generate CAR Tscm products.