Abstract
CAR-T, TIL, and TCR-based T cell immunotherapies have revolutionized cancer treatment; however, their clinical application remains limited by severe adverse effects such as CRS, ICANS, and HLH, as well as the risk of GvHD in the allogeneic setting. In contrast, NK cell-based therapies have emerged as a safer alternative with minimal adverse effects and can be developed from diverse cellular sources. Nevertheless, clinical translation of NK cell therapies is challenged by difficulties in large-scale expansion, donor variability, and high sensitivity to cryopreservation. In this study, we established a feeder-free culture system that selectively activated and expanded NK cells directly from peripheral blood mononuclear cells (PBMCs) without prior depletion of T or B cells. The expanded NK cells exhibited increased CD16 expression compared with naïve NK cells and demonstrated enhanced antitumor activity through antibody-dependent cellular cytotoxicity (ADCC) when combined with trastuzumab. To overcome limitations in long-term storage, NK cells were cryopreserved using a formulation containing 5% dimethyl sulfoxide (DMSO) supplemented with sugars and albumin. Importantly, the in vivo antitumor efficacy of cryopreserved NK cells was found to be comparable to that of freshly expanded NK cells. These findings show that feeder-free expanded NK cells possess potent ADCC activity and that optimized cryopreservation strategies can preserve therapeutic efficacy, supporting their clinical applicability and scalability as not only off-the-shelf allogeneic products but also personalized autologous NK cell therapies.