TY - JOUR T1 - Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression JF - bioRxiv DO - 10.1101/2021.08.06.455489 SP - 2021.08.06.455489 AU - Katherine P. Mueller AU - Nicole J. Piscopo AU - Matthew H. Forsberg AU - Louise A. Saraspe AU - Amritava Das AU - Brittany Russell AU - Madeline Smerchansky AU - Lei Shi AU - Keerthana Shankar AU - Adeela Ali AU - Cicera R. Lazzarotto AU - Shengdar Q. Tsai AU - Christian M. Capitini AU - Krishanu Saha Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/12/29/2021.08.06.455489.abstract N2 - Background Chimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.Methods Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.Results In comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.Conclusions This proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors.Competing Interest StatementConflicts of interest: K.P.M., N.J.P., M.H.F., L.A.S., A.D., C.M.C. and K.S. are inventors on a patent application related to this manuscript. C.M.C. receives honoraria for advisory board membership for Nektar Therapeutics and Novartis. No other conflicts of interest are reported. ER -