Abstract LB030: Efficient feeder cell-free in vitro expansion of NK cells for CAR-NK therapies using human platelet lysate

Abstract Building on the successes and addressing the limitations of CAR-T cell therapy, researchers are now exploring the use of natural killer (NK) cells as a potentially stronger candidate for the CAR based platform. While T cells are part of the adaptive immune system and need to be exposed and selectively activated and enriched after contact with exogenous or abnormal antigens in the body, NK cells are part of the innate immune system and exhibit inherent anti-tumor activity without requiring prior sensitization, making them attractive targets for CAR engineering (CAR-NK cells). By combining the potent cytotoxicity of NK cells with the specificity of CARs, CAR-NK therapies aim to provide a safer and potentially more broadly applicable form of cellular immunotherapy. In the last 5 years, there have been over 50 worldwide clinical trials registered in ClinicalTrials.gov using CAR-NK cells. The data shows that the use of CAR-NK cells leads to a lower incidence and severity of the cytokine release syndrome (CRS), due to the different nature of the activation and signaling pathways, compared to T cells. Additionally, NK cells offer less obstacles than T cells for the development of allogeneic “off the shelf” approaches, which would result in a more consistent and reliable therapy. Despite the higher safety and efficiency profile of the CAR-NK in comparison with CAR-T, there are some limitations that impede the further and faster advance of CAR-NK therapy over CAR-T therapy. The main limitation is the difficulty of obtaining clinically relevant numbers of CAR-NKs. NK cells are much less abundant that T cells, since they constitute just an average of 15% of the total peripheral blood lymphocytes. Additionally, NK cells have a much shorter life span and lower expansion capabilities in vitro. The efficient expansion of isolated NK cells in order to produce significant yields of CAR-NKs, currently depends on the co-culture with a feeder cell line for the activation and proliferation of NK cells. Even when the feeder cells (normally from tumor cell line) are used to promote NK proliferation, the efficiency and success rates are often low and variable patient to patient. Besides the low efficiency rates, the use of a live tumor cell line on the production process constitutes its own challenge in technical and regulatory aspects. We developed a protocol that involves the use of human platelet lysate (hPL) for in vitro maintenance, manipulation and expansion of human NK cells that, without the need of co-culturing with feeder cells of any type, enables robust in vitro expansion of human NK cells, surpassing the yields achieved with current state-of-the-art methodologies. NK cells expanded in vitro using hPL maintained their phenotypic and functional characteristics, as demonstrated through flow cytometry and tumor cytotoxicity assays, respectively. Citation Format: Vanesa Alonso-Camino, Andrew Sullivan, William Mirsch. Efficient feeder cell-free in vitro expansion of NK cells for CAR-NK therapies using human platelet lysate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB030.