Development of CAR NK Cell Lines Selectively Targeting Cancer Cells Expressing Membrane Hsp70

ABSTRACT An effective chimeric antigen receptor (CAR)‐based immunotherapy depends on both a suitable immune cell platform and a tumor‐specific antigen to overcome barriers in solid tumors. Natural killer (NK) cell lines are promising platforms for CAR constructs due to their inherent tumor‐killing ability, safety profile, and feasibility for standardized, off‐the‐shelf therapeutic use. Herein, four human NK cell lines (YT, KHYG1, NKL, and NK92) were retrovirally transduced with an anti‐Hsp70 CAR targeting membrane‐bound heat shock protein 70 (mHsp70), a tumor‐specific antigen with broad expression on many solid tumors, but not normal cells. Computational modeling suggested a strong binding between the CAR and the extracellular domain of mHsp70. Although all NK cell lines exhibited successful CAR integration and surface expression, only NKL and NK92 cells maintained stable CAR expression and long‐term viability. The anti‐Hsp70 CAR NKL and NK92 cells demonstrated enhanced expression of activation markers and secretion of cytotoxic effector molecules, and robust target‐specific killing of mHsp70‐positive cancer cells, while sparing mHsp70‐negative targets. Our findings validate the therapeutic potential of anti‐Hsp70 CAR NK cells and the suitability of NKL and NK92 cells for advancing off‐the‐shelf CAR NK cell therapies, thereby offering a promising strategy for targeting a broad range of solid tumors expressing mHsp70.