Integration of <i>ζ</i>-deficient CARs into the <i>CD3</i> <i>ζ</i> gene conveys potent cytotoxicity in T and NK cells
Jonas Kath, Clemens Franke, Vanessa Drosdek, Weijie Du, Viktor Glaser, Carla Fuster‐García, Maik Stein, Tatiana Zittel, Sarah Schulenberg, Caroline E. Porter, Lena Andersch, Annette Künkele, Joshua Alcaniz, Jens Hoffmann, Hinrich Abken, Mohamed Abou‐El‐Enein, Axel Prüß, Masataka Suzuki, Toni Cathomen, Renata Stripecke, Hans‐Dieter Volk, Petra Reinke, Michael Schmueck‐Henneresse, Dimitrios L. Wagner
Abstract
ABSTRACT: Chimeric antigen receptor (CAR)-redirected 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 nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. 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 human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection 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 to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection 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.