CAR NK-92 cell-mediated depletion of residual TCR+ cells for ultra-pure allogeneic TCR-deleted CAR T-cell products.

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 TCRα/β- 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 TCRα 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.