Non-viral TcBuster transposon engineering of CD70-CAR natural killer cells for the treatment of osteosarcoma.

Osteosarcoma (OSA) is the most common primary bone tumor in children and adolescents, yet outcomes have remained largely unchanged for over 40 years. While chimeric antigen receptor (CAR) T cell therapy has shown success in blood cancers, it faces major limitations in solid tumors due to immune evasion, antigen loss, and immunosuppressive tumor microenvironments. Natural killer (NK) cells offer several advantages over T cells, including multiple killing mechanisms and lower risks of graft-versus-host disease, neurotoxicity, and cytokine release syndrome, making them promising candidates for off-the-shelf cell therapies. However, unmodified NK cells have shown limited efficacy in clinical settings due to poor engraftment, persistence, and tumor-mediated suppression. To overcome these barriers, we developed a cost-effective method to engineer CAR NK cells targeting CD70, a tumor antigen overexpressed in relapsed and metastatic OSA. We further enhanced these cells by incorporating soluble interleukin-15 (IL-15) and a dominant-negative TGF-β receptor, creating "armored" CAR NK cells. These engineered cells resist transforming growth factor β (TGF-β) suppression, secrete IL-15, and demonstrate improved cytotoxicity, persistence, and tumor homing in both in vitro and in vivo models. Our findings support CD70 CAR NK cells as a promising immunotherapeutic strategy for relapsed and metastatic OSA.