Graft-versus-host disease (GvHD) is a major risk upon administration of allogeneic Chimeric Antigen Receptor (CAR) redirected T cells to HLA-unmatched patients. Gene editing can be used to disrupt potentially alloreactive T cell receptors (TCRs) in CAR T cells and reduce the risk of GvHD. Despite the high knock-out rates achieved with optimized methods, a subsequent purification step is necessary to obtain a safe allogeneic product. To date, magnetic cell separation (MACS) has been the gold standard to purify TCR_/_- CAR T cells, but product purity can still be insufficient to prevent GvHD. We have developed a novel, highly efficient approach to eliminate residual TCR/CD3+ T cells after TCR_ constant (TRAC) gene editing by adding a genetically modified CD3-specific CAR NK-92 cell line during ex vivo expansion. Two consecutive co-cultures with irradiated, short-lived, CAR NK-92 cells allow the production of TCR- CAR T cells with less than 0.01% TCR+ T cells, marking a 45-fold reduction of TCR+ cells compared to MACS-purification. Through an NK-92 cell-mediated feeder effect and by circumventing MACS-associated cell loss, our approach increases the total TCR- CAR T cell yield approximately 3-fold, while retaining cytotoxic activity and a favorable T cell phenotype. Scaling in the semi-closed G-Rex¨ bioreactor device provides proof-of-principle for large-batch manufacturing to allow for an improved cost-per-dose ratio. Overall, this cell-mediated purification method has the potential to advance the production process of safe off-the-shelf CAR T cells for clinical applications.

Author Info: (1) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (2) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (3) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (4)

Author Info: (1) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (2) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (3) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (4) Cellular Immunotherapy Program, Cancer Center, Massachusetts General Hospital, United States. (5) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (6) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (7) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (8) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany. (9) Berlin Institute of Health, Germany. (10) Berlin Institute of Health, Berlin, Germany. (11) CharitŽ - UniversitŠtsmedizin Berlin, corporate member of Freie UniversitŠt Berlin and Humboldt-UniversitŠt zu Berlin, Berlin, Germany. (12) Institute of Medical Immunology, Campus Virchow-Klinikum, CharitŽ - UniversitŠtsmedizin Berlin, Germany. (13) Keck School of Medicine, University of Southern California, Los Angeles, California, United States. (14) CharitŽ - UniversitŠtsmedizin Berlin, Berlin, Germany.