Enhanced feeder-free expansion, survival, potency and post manufacture stability of NK cells for CAR-NK therapies using human platelet lysate

Natural killer (NK) cells have emerged as a promising platform for chimeric antigen receptor (CAR) engineering, offering inherent anti-tumor activity and a favorable safety profile compared to CAR-T cells. Unlike T cells, NK cells belong to the innate immune system and do not require prior antigen sensitization, making them attractive for “off-the-shelf” allogeneic therapies. Clinical interest in CAR-NK cells has surged, with over 50 trials registered globally, driven by their reduced risk of cytokine release syndrome and graft-versus-host disease. Despite these advantages, the widespread adoption of CAR-NK therapies faces two major challenges: achieving clinically relevant NK cell numbers and ensuring post-manufacturing stability. NK cells are less abundant than T cells, exhibit limited in vitro expansion capacity, and have a short lifespan. Traditional methods rely on feeder cell co-culture, often using tumor-derived lines, which introduces technical and regulatory hurdles and yields inconsistent results. To address these limitations, we developed a feeder-free expansion protocol using human platelet lysate (hPL), enabling robust NK cell proliferation while preserving phenotype and cytotoxic function. Beyond expansion, we focused on a critical bottleneck in CAR-NK manufacturing-cryopreservation. Using proprietary cryopreservation solutions and protocols following hPL-based culture, we achieved significantly improved NK cell survival, maintenance of activating receptor expression, and retention of anti-tumor potency post-thaw. This advancement mitigates the logistical challenges of CAR-NK production, supporting scalable, ready-to-use cell therapy products. Our integrated approach-combining efficient feeder-free expansion with optimized cryopreservation-represents a major step toward reliable, high-quality CAR-NK therapies for clinical application.