Abstract 890: NK-92® (aNK™) whole cell lysate exerts potent cytotoxic and anti-proliferative activity on tumor cells
Authors: Himani Chinnapen, Laurent Boissel, Thomas Bickett, Courtney Fleenor, Vidya Godbole, Manju Saxena, Patrick Soon-Shiong, Hans Klingemann
Journal: Cancer Research (2025)

Lysates from human cells represent biofluids that are used in the biotechnology field for a number of applications, such as biomarker identification and antibody detection. Lysate from human blood platelets is widely used in the clinical setting to control bleeding. We hypothesized that whole cell lysate prepared from the natural killer cell line NK-92® or ‘activated’ NK (aNK™) has the potential as an immunotherapy modality for local treatment of cancer lesions, as it would be expected to contain an abundance of cytotoxic granule-associated serine proteases (granzymes), the pore-forming cytolytic protein (perforin), as well as an array of chemokines and cytokines that could contribute to a more immune-active tumor microenvironment. Here, we have generated whole cell lysate from NK-92 cells, and its high-affinity CD16 Fc receptor (158V) and erIL-2 -engineered variant haNK® cells, by repeated freeze/thaw cycles at a density of 108 cells/mL in a balanced salt solution (BSS) buffer containing no detergent, protease inhibitors, or EDTA. The resulting whole cell lysates were characterized for composition and cytotoxic activity. Western blot analysis confirmed the presence of perforin, granulysin, exosomal markers, and the full spectrum of granzymes known to be expressed in NK-92 cells. Biomarker profiling by Meso Scale Discovery-based assays revealed the presence of various chemokines and cytokines at significant concentrations in both NK-92 and haNK lysates, notably IL-2 (haNK only), IL-8, IL-10, IL-16, TNF-β, IFN-ɣ, and MIP-1α and β. A fluorometric assay confirmed enzyme activity of Granzyme B was maintained before and after cryopreservation of the lysate. Incucyte® SX3 live cell imaging assays showed that fresh lysate prepared from haNK cells displays cytotoxic and anti-proliferative activity against human and canine cancer cell lines after only a 15-minute exposure in vitro, suggesting cross-species specificity. Importantly, under the same conditions the lysate had no effect on primary human or canine cells. Intra-tumor injection of cryopreserved haNK lysate into intradermal tumors in immunocompetent C57BL/6 mice provided tumor control and clearance in two-thirds of the animals. Strikingly, when re-challenged with the same tumor cells 6-8 weeks later, no tumor growth occurred suggesting a vaccine-like effect of the haNK lysate. In conclusion, we have developed a simple, scalable method to prepare bioactive cell lysates from NK-92 and haNK that show anti-tumor activity and a potential for immune modulation both in vitro and in vivo. Citation Format: Himani Chinnapen, Laurent Boissel, Thomas Bickett, Courtney Fleenor, Vidya Godbole, Manju Saxena, Patrick Soon-Shiong, Hans Klingemann. NK-92® (aNK™) whole cell lysate exerts potent cytotoxic and anti-proliferative activity on tumor 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 890.