Expanded NK and fratricide-resistant CD70 CAR-NK cells are potent against patient-derived glioblastoma cells in vitro and in vivo

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by pervasive tumor recurrence and very poor patient survival. While αβ T cell-based chimeric antigen receptor (CAR) immunotherapies show efficacy in hematologic malignancies, manufacturing delays and potential fatal immune overactivation have prompted the exploration of alternative cell sources, including natural killer (NK) cells. NK cells represent an emerging cell source due to their potent antigen-independent intrinsic cytotoxicity, self-regulating inhibitory mechanisms, and low risk of graft-versus-host disease. In this study, we expanded peripheral blood NK cells using K562 feeder cells expressing membrane-bound IL-21 (K562-mb-IL-21) to produce highly activated and metabolically robust cells targeting both cell lines and patient-derived GBM tumor cells. Furthermore, our in vitro results demonstrate the broad targeting potential of our expanded NK cells, as they exhibit cytotoxicity against a cell line and patient-derived sample of another aggressive brain tumor, medulloblastoma (MB). To enhance tumor-directed cytotoxicity, we utilized CRISPR/Cas9 and adeno-associated virus-based gene delivery to generate stable anti-CD70 CAR-NK cells. CD70 is a promising target due to its minimal expression in healthy tissues and overexpression in recurrent GBM. However, NK cells gain CD70 surface expression upon expansion, causing anti-CD70 CAR-NK cell fratricide. Subsequently, knocking out the CD70 gene enabled large-scale expansion of anti-CD70 CAR-NK cells that were functional against both in vitro and in vivo GBM models.