Cord Blood‐Derived Fibroblast Activation Protein‐Targeted Chimeric Antigen Receptor Natural Killer Cells Persist in Vivo

Abstract

ABSTRACT Cancer‐associated fibroblasts (CAFs) have been recognized as key contributors to tumor progression. Chimeric antigen receptor (CAR) T‐cell therapy targeting fibroblast activation protein (FAP), a marker of CAFs, has gained attention and is being evaluated in both preclinical and clinical studies. Cord blood (CB)‐derived CAR natural killer (NK) cell therapy has shown efficacy in hematologic malignancies. In this study, we generated FAP‐targeting CAR NK cells (FAP‐CAR NK cells). CD3‐depleted CB mononuclear cells were stimulated with irradiated K562 cells expressing 4‐1BB ligand (tumor necrosis factor ligand superfamily member 9) and membrane‐bound interleukin‐15 and interleukin‐21 to expand NK cells, into which FAP‐CAR was introduced. FAP‐CAR NK cells produced cytokines and exerted cytotoxic activity when co‐cultured with FAP‐transduced HT1080 cells and CAFs isolated from human lung cancer tissue. In a xenograft model established by intrathoracic co‐injection of A549 lung cancer cells and luciferase‐expressing CAFs, injection of FAP‐CAR NK cells, but not control CD19‐CAR NK cells, resulted in their robust expansion and persistence in vivo for 5 weeks. However, CAFs were not eliminated by FAP‐CAR NK cells, although CAR NK cells were clearly detected in the tumors. The levels of cytotoxicity‐associated molecules (such as granzymes) were higher in FAP‐CAR NK cells persisting in the spleens of mice compared with those in pre‐infusion FAP‐CAR NK cells. These results indicate that FAP‐CAR NK cells have the potential to respond specifically to the target antigen and expand robustly in vivo, although further optimization is required to maintain cytotoxic function for a prolonged duration in vivo.