Anti-CD19 CAR-NK cells coexpressing GITRL to enhance antitumor activity for the treatment of leukemias and lymphomas.

In vivo assay in a murine leukemia model was performed to evaluate tumor burden and survival. CAR19-GITRL-NK-92 cells exhibited greater expansion capacity, a more active metabolic profile (glycolysis and ATP production), in addition to increased expression of NKp44 and NKp46. Both CAR cells exhibited cytotoxicity nearly 40% higher than that of NK-92 cells, accompanied by increased cytokine secretion (TNF-α and IFN-γ) and degranulation.

Although the presence of GITRL did not directly enhance tumor cell lysis in vitro, its expression was upregulated following CAR activation by target cells. RNA-Seq analyses revealed activation of pro-effector pathways (KIT, IRF5) and downregulation of inhibitory signals (IL1RN). For the in vivo assay, CAR19-GITRL-NK-92 cells exhibited enhanced tumor control and prolonged survival.

In primary NK cells, coexpression of GITRL increased in vitro cytotoxicity and conferred partial resistance to Treg-mediated suppression, with a 43% reduction in growth, compared to 79% observed in CAR19-PB-NK cells. In conclusion, GITRL coexpression in CAR19-NK cells led to a more active metabolism, enhanced activation of pro-cytotoxic pathways, and superior tumor control in a murine model, as well as partial resistance to Treg-mediated immunosuppression in primary cells.

Future studies, particularly in vivo, will be essential to validate and extend these findings. These results emphasize the translational potential of CAR19-GITRL-NK cells as an innovative strategy for the treatment of B-cell leukemias and lymphomas.