Integration of ζ-deficient CARs into theCD3-zetagene conveys potent cytotoxicity in T and NK cells

Abstract

I.AbstractChimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the humanCD3ζ(CD247)gene, functional CAR fusion-genes are generated that exploit the endogenousCD3ζ gene as the CAR’s activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells,CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings.CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control toT cell receptor alpha constant(TRAC)-replaced and lentivirus-transduced CAR-T cellsin vivo. Tuning ofCD3ζ-CAR-expression levels significantly improved thein vivoefficacy. Compared toTRAC-edited CAR-T cells, integration of a Her2-CAR intoCD3ζ conveyed similarin vitrotumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably,CD3ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus,CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.Key pointsIntegration of ζ-deficient CARs intoCD3ζ gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf useCD3ζ-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions