Abstract 894: The race is on: BiTE vs CART targeting FLT3 in AML
Authors: Rohrbacher Lisa , Nixdorf Daniel , Stadler Helena , Brauchle Bettina , Märkl Florian , Philipp Nora , Hänel Gerulf , Marcinek Anetta , Kazerani Maryam , Goldstein Rebecca L. , von Bergwelt Michael , Kobold Sebastian , Bücklein Veit L. , Arvedson Tara , Subklewe Marion 
Published: April 2023

Abstract
Translation of the success of bispecific T cell engagers (BiTE®) and CAR T cells from B-cell to myeloid malignancies has been challenging. Identifying suitable target antigens in myeloid malignancies has been hampered by expected on-target-off leukemia toxicity. FLT3 is a promising target antigen with high expression in most AML samples independent of FLT3 mutation status (Brauchle et al., 2020). Here, we compared two T-cell based immunotherapy approaches, BiTE® vs CART, in targeting FLT3 in AML. We tested BiTE® and CAR mediated cytotoxicity against several AML cell lines and primary AML (pAML) cells in vitro. The impact of positive co-stimulation was evaluated using our previously established Ba/F3 model system, which is devoid of human costimulatory molecules. Conjugate formation as a surrogate for synapse formation and consecutive T-cell degranulation were analyzed. Combination with tyrosine kinase inhibition (TKI) was tested in conjunction with BiTE® and CAR to further enhance cytotoxicity against FLT3mut AML cells. On-target off-leukemia toxicity was tested in mixing assays of AML cells and healthy bone marrow (hBM). Finally, we compared CAR and BiTE® in an in vivo xenograft AML model. No differences in BiTE® vs CAR mediated cytotoxicity were observed against cell lines with different genetic background and FLT3 expression levels or pAML samples. However, the percentage of T cell-AML conjugates formed utilizing CART was significantly higher compared to BiTE® molecules. Also, faster and stronger degranulation of CD107a was detected using CAR T cells vs BiTE®. Interestingly, we showed that expression of the costimulatory CD86 increased the cytotoxicity of BiTE®-redirected T cells while CAR mediated lysis was unaltered. In case of the FLT3-ITD mutated cell line MV411, the addition of FLT3 TKIs significantly increased CAR and BiTE® mediated cytotoxicity. Looking at on-target off-leukemia toxicity, in particular hBM cells, we only observed low cytotoxicity. Moving to an in vivo AML xenograft model, we observed that CAR T cell treated mice had an improved OS compared to BiTE®-treated mice, accompanied by enhanced proliferation and homing of the CAR T cells to the spleen. We conclude, that FLT3 is a promising target antigen for T cell-based immunotherapy with limited on-target-off leukemia toxicity. Our preclinical in vitro data demonstrate similar cytotoxicity of both platforms against AML cell lines and pAML cells. Utilizing a xenograft mouse model, CAR T cells provided a significant survival benefit over BiTE® molecules. Our data support the hypothesis that positive co-stimulation either integrated within the construct (e.g. 2nd generation CAR) or provided by the target cell (e.g. CD86 expressing leukemia cells) propagates Tcell activation and proliferation. Future studies will be needed to further dissect differences between BiTE® vs CAR T cell based immunotherapy platforms and identify suitable combination partners.
Citation Format: Lisa Rohrbacher, Daniel Nixdorf, Helena Stadler, Bettina Brauchle, Florian Märkl, Nora Philipp, Gerulf Hänel, Anetta Marcinek, Maryam Kazerani, Rebecca L. Goldstein, Michael von Bergwelt, Sebastian Kobold, Veit L. Bücklein, Tara Arvedson, Marion Subklewe. The race is on: BiTE vs CART targeting FLT3 in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 894.