As Fast As We Can: Rational Design of Rapid Chimeric Antigen Receptor T Cell Manufacturing

Abstract

Background Chimeric antigen receptor T cell proved to be a promising approach to treat hematological malignancies. There are six autologous CAR-T drugs approved by FDA in US. The production of autologous CAR-T cells is carried out by a time-consuming manufacture process typically need 2-3 weeks. Unfortunately, about 25% patients died while awaiting their CAR-T product. Rapid manufacturing CAR-T cells still is an unmet clinical need for fast processing diseases. We developed the “DASH CAR-T” manufacturing platform which harvested CAR-T cell in two-three days. Our first product, autologous DASH CD19 CAR-T demonstrated greater anti-tumor efficacy in murine study correlated with higher T cell expansion compared with conventional CAR-T. In this study, we designed a simplified novel CAR-T manufacturing process with optimized culture and harvest time between 48 hours to 72 hours. Methods Rapid manufacturing CAR-T cells comprising common key steps as the conventional CAR-T product : select T cells from PBMC, activated T cells by CD3/28 Dynabeads, transduced with retroviral vector expressed CD19-28z CAR construct, and then the CAR-T cells harvested and cryopreserved for clinical use. We investigated the shortest time for each steps by various reagents, medium and process. Then we compressed the whole process and our novel approaches completed all manufacturing process less than 72 hours. Results First, we compared the harvest timing based 3 batches CAR-T cells from health donors. A various factors, such as cell viability, CD3+%, Annexin V-/PI- population, percentage of residual CD19+ cells, and naïve T cell proportion were very similarly between 48 hours CAR-T cells and 72 hours CAR-T cells, indicated comparable harvest timing for manufacture. Second, we investigated 3 batches of CAR-T cells manufactured from B cell malignancy patients. In this scenario, 72 hours CAR-T exhibited better cell viability, increased naïve T cell population, and more Annexin V-/PI- cells in the culture compared to 48 hours CAR-T. The different performance of patient cells and healthy donor cells suggested the importance of the choice of starting material for process development of cell therapy. All batches successfully manufactured in 72 hours. Tumor cell-xenograft NOG mice models were confirmed the anti-tumor activity of DASH CD19 CAR-T cells in vivo. Conclusions This study reported an optimized DASH CAR-T process with 48 hours and 72 hours manufacturing. Cell viability, stemness of T cells, CAR expression, and removal of impurity were improved in the CAR-T product manufactured by this novel process. Based on the preclinical study results, an investigator-initiated trial (IIT) has launched to explore the safety, efficacy, pharmacokinetic/pharmacodynamics characteristics in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL).