Abstract 894: Generation of cytokine-induced memory-like PD-L1 t-haNK (NK-92®) cells

Abstract Cytokine-induced memory-like NK cells have garnered a lot of attention as cellular therapeutic agents due to their much-increased lifespan in patients and their enhanced memory response upon re-challenge by cytokines or target cells. A key part of this memory recall feature is the increased secretion of interferon gamma (IFNγ). This potent immune stimulator can trigger upregulation of MHC-I molecules expression on the surface of tumor cells, thereby pushing the tumor towards an immunologically “hot” status. However, IFNγ also triggers expression of the immune checkpoint molecule PD-L1. We show here that the Natural Killer cell line NK-92®, or ‘activated’ NK (aNKTM), can acquire a memory-like phenotype upon overnight induction with IL-12, IL-18, and the IL-15 superagonist N-803, as evidenced by increased steady-state IFNγ secretion (>50-fold increase) and up-regulation of CD25. Although the initial increase in IFNγ secretion subsides after 48hrs, memory-like NK-92 cells display memory recall for several days post-activation, as evidenced by an enhanced IFNγ response upon challenge with K562, Raji, and THP-1 cells compared to non-memory-like cells (2.3, 6.9, and 3.8-fold increases after 1 week, respectively). Further, NK-92 cells engineered to express a high-affinity Fc receptor and an anti-PD-L1 Chimeric Antigen Receptor (PD-L1 t-haNK) maintained their susceptibility to memory-like cytokine induction. Exposure of the NK-resistant cells lines THP-1 and SK-ES-1 to culture supernatant from memory-like PD-L1 t-haNK triggered expression of PD-L1 on their surface, rendering them highly sensitive to PD-L1 CAR-mediated targeted killing by the PD-L1 t-haNK cells in a 4-hour in vitro cytotoxicity assay (respectively 91% and 88% killing at E:T ratio of 1.25:1, compared to 54% and 41% for target cells exposed to regular PD-L1 t-haNK supernatant), while not affecting their resistance to NK-92 cells. Infusion of a cellular therapy product that constitutively secretes high IFNγ may cause detrimental side effects. Memory-like PD-L1 t-haNK cells may circumvent this problem through their memory recall ability and deliver increased amount of IFNγ specifically at the tumor site when locally engaging with tumor cells. This increase would then drive upregulation of PD-L1 expression on neighboring tumor cells, creating an expanding wave of targets sensitized to killing by PD-L1 t-haNK cells. A next generation, memory-like PD-L1 t-haNK cell line equipped with inducible safety switch features to remove the need for irradiation and extend their half-life in patients is being developed and would constitute a promising “off-the-shelf” cellular therapy against immunologically cold cancers. Citation Format: Dustin Elwood, Devapriya Segaran, Joseph Thibault, Vidya Godbole, Manju Saxena, Hans Klingemann, Laurent Boissel, Patrick Soon-Shiong. Generation of cytokine-induced memory-like PD-L1 t-haNK (NK-92®) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 894.