Immune cells reprogrammed with Chimeric Antigen Receptor (CAR) technology offer substantial therapeutic promise in the fields of oncology, autoimmune disorders, transplant medicine, and infectious diseases. Traditional CAR-T cell therapies rely on autologous manufacturing using undirected viral gene transfer, resulting in logistical challenges and high costs per treatment. Further, viral vectors induce unphysiological regulation of CAR expression and increase the risk for malignant transformation through insertional mutagenesis of proviruses. To overcome these challenges, we developed non-viral gene editing of the CD3ζ (CD247) gene to reprogram immune cells for adoptive transfer applications.

By integrating truncated CAR-transgenes devoid of a primary activation domain into the CD3ζ-gene, we create functional CAR fusion-genes. This strategy harnesses the endogenous CD3ζ-gene as the CAR's activation domain, ensuring physiological regulation of CAR expression across various immune cell types including conventional T cells, TCRγδ T cells, regulatory T cells, and NK cells. Notably, this CD3ζ in-frame fusion approach also eradicates TCR surface expression in T cells, significantly reducing the risk of graft-versus-host disease in allogeneic off-the-shelf applications.

Our results demonstrate that CD3ζ-CD19-CAR-T cells exhibit leukemia control comparable to both TRAC-replaced and lentivirus-transduced CAR-T cells in vivo. Furthermore, tuning the expression level of the CD3ζ-CAR enhances its in vivo efficacy. Compared to CD19-specific TCR-like CAR architectures (HIT/STAR, eTruC receptor design), our CD3ζ-CAR T cells displayed superior functionality in vivo. Remarkably, CD3ζ-gene editing also successfully reprograms NK cells without compromising their intrinsic functions. In conclusion, CD3ζ-gene editing presents a promising and versatile platform for developing allogeneic off-the-shelf cell therapies utilizing redirected killer lymphocytes.