W194 - Precise Integration of Truncated Cars into the cd3-zeta Gene Conveys Potent Cytotoxicity in T and NK Cells

Clemens Franke; Vanessa Drosdek; Weijie Du; Viktor Glaser; Maik Stein; Tatiana Zittel; Lena Andersch; Annette Künkele; Renata Stripecke; Hans-Dieter Volk; Petra Reinke; Michael Schmueck-Henneresse; Dimitrios Laurin Wagner

Abstract Text: The use of chimeric antigen receptor (CAR)-reprogrammed immune cells through adoptive transfer has the potential to revolutionize oncology, autoimmune diseases, and transplant medicine. However, the current reliance on personalized cell manufacturing using viruses as vectors for gene transfer makes these therapies difficult to access due to logistical challenges and high costs. One promising way to overcome these limitations is virus-free gene editing, which allows for precise engineering of safe CAR immune cells without alloreactivity, enabling large-scale production of allogeneic CAR products. In this study, we transfer truncated CAR-transgenes not encoding a main activation domain into the human CD3ζ (CD247) gene using CRISPR-Cas. This creates functional CAR fusion-genes that utilize the endogenous CD3ζ gene as the CAR’s activation domain. This procedure allows CAR-reprogramming of conventional T cells, γ/δ T cells, regulatory T cells and natural killer (NK) cells. In T cells, this strategy prevents T cell receptor (TCR) surface expression, thereby eliminating the risk of alloreactivity. CD3ζ-CD19-CAR T cells show better tumor control than lentivirus-transduced CAR T cells in vivo. Furthermore, CD3ζ-integration of a HER2-CAR conveys similar tumor control but with reduced susceptibility to activation-induced cell death in vitro compared to TCRα constant (TRAC)-edited T cells. In contrast to the TRAC approach, CD3ζ editing also enables reprogramming of NK cells to enhance their cytotoxicity in a CAR-dependent fashion and without affecting missing-self recognition or antibody-dependent cytotoxicity. Thus, our findings suggest that CD3ζ gene editing can serve as a universal platform technology to redirect killer lymphocytes for allogeneic off-the-shelf cell therapies.