Preparation of cryopreserved chimeric antigen receptor T cells for the locoreogional delivery to the neural axis

Background aims: Intracranial (IC) locoregional delivery of chimeric antigen receptor (CAR) T cells presents an attractive delivery method to central nervous system tumors. Although IC delivery is actively being employed in early-phase clinical studies, no thaw /wash methods have been published to remove the neurotoxic cryoprotectant dimethyl sulfoxide (DMSO) from CAR T-cell products before IC administration. Thus, the aim of this study was to develop and validate a simple thaw/wash procedure.Methods: We developed a thaw/wash procedure that consist of product thaw at 37(degrees)C, equilibration for 5 min in 1 volume of preservative-free normal saline (PFNS), dilution with an additional 8 volumes of PFNS, removal of DMSO through a washing step, resuspension in 2.0 mL of PFNS and storage in a syringe at 20-25(degrees)C. Final for-mulated products (FPs) were assessed for quality and safety attributes and stability over 3 h from the comple-tion of the thaw. Stability parameters included CAR T-cell viability, transgene surface expression and cytolytic activity.Results: The developed procedure reduced the calculated % of DMSO to less than 0.025%. FP cell viability and recovery (versus pre-cryopreservation) were within acceptable specifications (mean viability: 85.3%, range: 83%-88%; total nucleated cell recovery mean: 76.5%, range: 65.4%-82.5%). Other prespecified quality assur-ance/quality control parameters including appearance/ integrity, sterility and endotoxin level (<= 1.0 EU/mL), were also met by all FPs (n = 3). Three hours' post thaw/wash stability was confirmed. All products main-tained cell viability greater than 70% (mean, 80.0%; range, 79%-81%), with no significant change in transgene expression or cytolytic activity of B7-H3-CAR T cells compared with thawed not diluted/washed control CAR T cellsConclusions: We have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products.(c) 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.