Effective gmp-compliant point of care manufacturing of anticd19 chimeric antigen receptor t cells for non hodgkin lymphoma patients using the clinimacs prodigy®

Abstract

Background & Aim Chimeric Antigen Receptor T-Cells (CAR Ts) have shown promising results in clinical studies to treat lymphomas, however clinical scale GMP-compliant manufacturing of CAR T cells can be challenging and unreproducible. We successfully manufactured autologous lentiviral anti-CD19 Chimeric Antigen Receptor T Cells for 18 relapsed or refractory patients in an investigator initiated Phase I clinical trial using the CliniMACS Prodigy®. Methods, Results & Conclusion T-Cells were isolated, transduced (LV-anti-CD19 CAR, Lentigen Technology, Inc.), and expanded in culture for 8 or 12 days. All 18 cultures met cell dose and safety requirements. Fifteen cultures were expanded for 12 days, then the culture time was reduced to 8 days to facilitate treatment to patients in an expedited manner. The 12 day cultures had a mean transduction efficiency of 48± 0.1% (range 29-62), a mean of 42-fold expansion ± 12 (range 30-78), and the mean number of CAR T+ cells at the end of culture was 2 × 109± 0.8 × 109. The 8 day cultures had a mean transduction efficiency of 42%, a mean of 12 fold expansion, and the mean CAR T+ cells at the end of culture was .6 × 109 cells. There were no manufacturing failures. For all patients, a predominance of CD8 cells was observed in the leukapheresis and post enrichment product. However, the phenotype of the infused products had a 2 fold increase of CD4 CAR T+ cells: CD8 CAR T+ cells. CAR T products were infused fresh for 16 patients and 2 received cryopreserved products. While the frequency of CAR T+ cells was not affected by freeze thaw, a decrease in T-effector memory cells was observed after thaw. All cultures were transduced with an MOI of 20 and had a mean vector copy number of .9 copies/cell. Patients monitored post infusion for vector persistence through proviral qPCR and flow cytometry show peak expression of CAR T+ cells between 6 days and 21 days post infusion, with most patients peaking around 14 days post infusion. To date, detectable vector has persisted for up to 12 months. Lastly, our CAR T products were tested for potency and showed a dose-dependent cytotoxicity towards CD19+ cancer cell lines in vitro. In conclusion, GMP-compliant anti-CD19 Chimeric Antigen Receptor T Cells can efficiently be manufactured using the CliniMACS Prodigy in as few as 8 days, resulting in a potent cell product and an expedited treatment for patients.