Generation of allogeneic CAR-NKT cells from hematopoietic stem and progenitor cells using a clinically guided culture method

Abstract Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using ‘off-the-shelf’ products, such as allogeneic CAR natural killer T ( Allo CAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into Allo CAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced Allo CAR-NKT cells with high yield and purity. We generated Allo CAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of Allo CAR-NKT cells support their potential for clinical translation.