Abstract 3174: BiTE® molecule vs CAR-T targeting FLT3 in AML: How positive co-stimulation tips the scale

Abstract T cell-based immunotherapy has revolutionized the treatment of B-cell malignancies, yet applying it to acute myeloid leukemia (AML) is challenging due to difficulties in identifying suitable target antigens without on-target off-leukemia toxicity. Prior studies identified FLT3 as a promising target antigen with restricted expression in the healthy hematopoietic compartment1. Here, we evaluate a FLT3-directed BiTE® molecule and 2nd generation FLT3-specific CAR T cells in a preclinical AML model. We hypothesize that positive costimulatory molecules on AML cells enhance BiTE® molecule effectiveness, while CAR T cells may be less dependent due to their built-in costimulatory domain. Cytotoxicity was assessed in cocultures over time using MPFC. The impact of positive costimulation was tested using our established Ba/F3 model system lacking any human costimulatory molecules. On-target off-leukemia toxicity was evaluated in cytotoxicity and CFU assays of pAML cells and healthy bone marrow (hBM). T-cell exhaustion was examined in a longterm culture system with continuous antigen exposure. Lastly, xenograft AML models and RNA sequencing explored differences between the two platforms in vivo. BiTE® molecule and CAR effectively mediated cytotoxicity against various AML cell lines and pAML cells. Overexpression of the costimulatory CD86 on the target cells significantly increased BiTE® molecule-mediated cytotoxicity cells, while CAR-mediated lysis was unaltered. In both scenarios, low E:T-dependent toxicity was observed against hBM. Mixing experiments showed efficient pAML cell lysis with minimal impact on hBM. CFU assays indicated no effect on hematopoiesis of healthy CD34+ stem cells. 28-day cocultures led to T-cell exhaustion in both settings, marked by reduced proliferation, cytokine secretion, metabolic fitness, and cytotoxicity. CAR T cells demonstrated superior antileukemic activity, with enhanced proliferation and splenic homing in an AML xenograft model. RNA sequencing revealed more exhaustion-related markers in BiTE® molecule-redirected T cells, while CAR T cells showed upregulated glycolysis and fatty acid metabolism. Repeating our in vivo xenograft experiment with CD86-overexpressing AML cells supported our hypothesis that costimulation might be a key factor providing a head start to CAR T cells by increasing T-cell proliferation and persistence in vivo. Our in vitro data show similar cytotoxicity and specificity, while xenograft mouse models suggest CAR T cells offer a significant survival advantage over BiTE® molecules. Our data support the hypothesis that positive costimulation integrated within the CAR constructs boosts T-cell activation, homing, and efficacy, thereby delaying T-cell exhaustion in vivo. Future studies are needed to further dissect differences between BiTE® molecule vs CAR T cell-based immunotherapy and identify suitable patient groups. 1Brauchle et al 2020 Citation Format: Lisa Rohrbacher, Daniel Nixdorf, Helena Stadler, Bettina Brauchle, Florian Märkl, Adrian Gottschlich, Gordon Hoffmann, Nora Philipp, Gerulf Hänel, Martin Kirmaier, Anetta Marcinek, Maryam Kazerani, Alica-Joana Emhardt, Giulia Magno, Rebecca L. Goldstein, Sebastian Theurich, Tobias Straub, Sebastian Kobold, Tara Arvedson, Veit L. Bücklein, Marion Subklewe. BiTE® molecule vs CAR-T targeting FLT3 in AML: How positive co-stimulation tips the scale [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 3174.