Preclinical assessment of HER2 CAR-T cells using tumor and GI organoid models to define therapeutic window

Anti-HER2 therapies have significantly improved outcomes for metastatic breast cancer patients with HER2+ tumors. Beyond antibody-based approaches such as trastuzumab, HER2-targeted CAR-T cells are emerging as promising options for solid tumors. However, the clinical translation of cancer therapies is often hindered by off-target toxicities, particularly in the gastrointestinal (GI) tract. CAR-T cells, can cause epithelial damage, leading to adverse events such as diarrhea and mucosal inflammation. To address this, we employed 3D iPSC-derived intestinal organoids (HIOs) as a human physiologically relevant model to assess GI toxicity. Using viability assays, cleaved caspase 3 and 7, we evaluated both direct cytotoxic and immune-mediated epithelial injury in HIOs co-cultured with PBMCs. In parallel, we investigated the efficacy of HER2-targeting CAR-T cells versus trastuzumab as positive control in HER2+ and HER2− human cancer cell lines (SK-OV3, JIMT-1, Hs578T) in 2D and 3D in vitro models. Tumor growth and invasion of the CAR-T cells was measured via fluorescence-based live cell imaging. On the last experiment day, a metabolic read-out (CellTiter-Glo, CTG assay) was performed. CAR-T cells demonstrated potent, dose-dependent cytotoxicity against HER2+ spheroids, including trastuzumab-resistant JIMT-1, while sparing HER2− Hs578T cells. Trastuzumab was effective only in SK-OV3. The un-transduced T cells proved to be ineffective. In parallel, the CAR T cells induced epithelial injury in the HIOs indicating possible safety concerns in later drug development stages. Also, in this assay the untransduced T cells from the same donor did not induce any cytotoxicity. In summary, the integration of intestinal organoid-based toxicity screening with tumor efficacy assays enabled a more precise definition of the therapeutic window for HER2 CAR-T cells. This dual-platform approach enhances preclinical evaluation by simultaneously capturing efficacy and GI safety, supporting the development of cell therapies with improved translational potential.