Feeder-Free Expanded and Cryopreserved NK Cells Retain Antibody-Dependent Cell Cytotoxicity against HER2-Positive Tumors

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 <i>in vivo</i> 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.