Quintarelli et al. developed a feeder-cell-free NK cell manufacturing approach amenable to lentivirus transfection with CAR constructs to produce CD19 CAR-NK cells with high efficiency, maintaining a diverse differentiation pattern and low expression of PD-1. In vitro, CAR-NK cells were cytotoxic against multiple CD19-expressing leukemia lines, including primary ALL blasts. Expression of key NK activating receptors was not required for CAR-NK activity but was maintained, potentially mitigating antigen escape via CD19 loss. In vivo, CAR-NK cells demonstrated equivalent efficacy to CAR T cells against DAUDI tumors but without cytokine-mediated fatal toxicity.
We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19(+) cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.