114P Exploring the role of FES kinase in natural killer cells to enhance immunotherapy

Background: Cytokine release syndrome (CRS) and immune cell associated neurotoxicity syndrome (ICANS) are major toxicities associated with CD19-directed CAR Tcell therapies.These toxicities result from supraphysiologic release of inflammatory cytokines such as IL-6, IFN-, TNF-, and chemokines like MCP-1 and IP-10.While systemic corticosteroids or IL-6 receptor blockade can be used in the treatment of these toxicities, they may broadly suppress inflammatory signaling pathways placing vulnerable patient populations at risk of serious, life-threatening infections, steroid induced myopathies, or inferior outcomes.CTO1681 is a novel small molecule therapeutic designed to suppress CRS-driving cytokine production without compromising chimeric antigen receptor (CAR) T-cell function.This study evaluated the effect of CTO1681 on cytokine modulation and CAR T-cell-mediated tumor killing in vitro.Methods: Human CD19 CAR T-cells were co-cultured with Raji lymphoma cells for 24 hours at effector-to-target (E:T) ratios of 10:1, 5:1, and 1:1.CTO1681 was tested at doses ranging from 0.03 to 1 M alongside vehicle control.Inflammatory cytokines (e.g., TNF-, IL-6, IFN-) were assessed in culture supernatants using Luminex multiplexing assay.CD19 CAR T-cell mediated killing of Raji lymphoma cells was assessed using flow cytometric analysis of CD19 expressing cells.Results: CTO1681 induced a dose-dependent reduction in key CRS-associated cytokines.In the 10:1 cultures, treatment with 225 nM reduced TNF-, IL-6, and IFN- levels by 52%, 26%, and 16%, respectively, compared to untreated cells.Importantly, CTO1681 did not impair CAR T-cell-mediated cytotoxicity.Raji cell killing remained consistent across all doses of CTO1681, with killing titrated appropriately by E:T ratio: 25% at 10:1, 23% at 5:1, and 18% at 1:1.Conclusions: These findings support CTO1681 as a promising CRS-modulating agent that reduces inflammatory cytokines while preserving CAR T-cell function in vitro, warranting further development alongside CAR T-cell therapies.