Abstract 7274: A preclinical <i>in vitro</i> platform for evaluating the potency of bispecific T cell engagers in 2D and 3D models

Abstract Introduction: Bispecific T-cell engagers (BiTEs) are innovative cancer therapeutics designed to harness the immune system by linking T cells to cancer cells, inducing tumor-specific cytotoxicity, which enhances specificity and reduces off-target effects on normal tissue. In this study in vitro assays were established to evaluate the tumor cell-killing and T cell activation of BiTEs, specifically using a claudin18.2-targeted BiTE AMG910 and the monoclonal antibody zolbetuximab. Methods: Target expression on tumor cells for claudin18.2 and AMG910/zolbetuximab binding to tumor and T cells was evaluated via flow cytometry. Tumor cell killing was assessed after 48h co-culture of tumor cells with human T cells upon treatment by FACS and LDH release assays. T cell activation was evaluated by IFN-γ and IL-2 supernatant release, along with FACS (CD25, CD69, IFN-γ, and Granzyme B). AMG910 was further tested in 3D pancreatic patient-derived xenograft organoid (PDXO) models employing high content imaging (HCI) to assess immune cell activation, infiltration, and organoid killing. Results: NUGC4-hClaudin18.2 cells exhibited the highest Claudin18.2 protein levels compared to NUGC-4 and SNU-620 cells, despite similar mRNA levels. RNA-seq and FACS showed that Claudin18.2 expression was absent in AGS cells. AMG910 displayed stronger binding to SNU-620 cells and T cells compared to zolbetuximab, while zolbetuximab exhibited stronger binding to NUGC4-hClaudin18.2 cells, with no significant binding difference observed between the two Abs on NUGC-4 cells. In a co-culture with human T cells and tumor cells, AMG910 demonstrated significant dose-dependent killing of SNU-620, NUGC-4, and NUGC4-hClaudin18.2 cells compared to untreated samples, along with elevated IFN-γ and IL-2 secretion. T cell activation was further confirmed by increased expression of CD25, CD69 and intracellular Granzyme B and IFN-γ. No effects were observed in co-culture with AGS cells. Furthermore, AMG910 did not activate T cells in the absence of tumor cells, indicating AMG910’s Claudin18.2-dependent T cell activation. In 3D co-cultures, treatment with the T cell engager enhanced migration of activated immune cells (CD69) into the organoids, inducing organoid killing analyzed via HCI. Conclusion: Successful in vitro assays were developed for preclinical BiTEs testing. 2D co-cultures efficiently assess direct tumor cell killing and T cell activation mediated by antibody treatments. Additionally, 3D organoid co-cultures offered a more physiologically relevant setup, mimicking the tumor microenvironment and representing patient diversity. Moreover, the HCI analysis revealed enhanced infiltration of activated T cells upon treatment with the T cell engagers before killing of the organoids. This combination offers a robust platform for in-depth preclinical evaluation of BiTEs. Citation Format: Xuefei Yan, Shiqi Guo, Saskia De Man, Yifan Chen, Jiawei Meng, Michelle Kop, Valeria Teodosieva, Haijuan Yu, Gera Goverse, Jun Zhou, Peng Wang. A preclinical in vitro platform for evaluating the potency of bispecific T cell engagers in 2D and 3D models [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 7274.