Depletion of an immature cord blood NK subset reverses trogocytosis-driven CAR NK dysfunction.

Allogeneic CAR-engineered NK cells enable scalable off-the-shelf therapy, yet donor heterogeneity remains a major barrier to consistent potency. Building on our clinical experience with cord blood-derived CAR NK cells, we identified an immature CD16-CD161- double-negative (DN) NK subset associated with poor outcomes. Following CAR engineering, DN-derived NK cells remained hypofunctional yet exhibited exaggerated trogocytosis, accumulating high levels of cognate antigen and forming an intra-product trogocytic-antigen sink (TAS) that diverted CAR engagement from tumor targets and promoted on-target off-tumor interactions within the product. Consequently, CD16+CD161+ double-positive (DP) effectors exposed to the TAS underwent sustained activation, metabolic stress, fratricide, reduced viability, programmed cell death, and progressive exhaustion, culminating in impaired persistence and tumor control. Pre-manufacturing DN depletion eliminated the TAS and restored DP antitumor activity across hematologic and solid tumor models. Together, these findings define a donor subset-driven mechanism of CAR NK dysfunction and provide a manufacturing strategy to improve antitumor potency.