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T cell bispecific antibodies (TCBs) are the focus of intense development for cancer immunotherapy. Recently, peptide-MHC (major histocompatibility complex)-targeted TCBs have emerged as a new class of biotherapeutics with improved specificity. These TCBs simultaneously bind to target peptides presented by the polymorphic, species-specific MHC encoded by the human leukocyte antigen (HLA) allele present on target cells and to the CD3 coreceptor expressed by human T lymphocytes. Unfortunately, traditional models for assessing their effects on human tissues often lack predictive capability, particularly for "on-target, off-tumor" interactions. Here, we report an immune-infiltrated, kidney organoid-on-chip model in which peripheral blood mononuclear cells (PBMCs) along with nontargeting (control) or targeting TCB-based tool compounds are circulated under flow. The target consists of the RMF peptide derived from the intracellular tumor antigen Wilms' tumor 1 (WT1) presented on HLA-A2 via a bivalent T cell receptor-like binding domain. Using our model, we measured TCB- mediated CD8+ T cell activation and killing of RMF-HLA-A2-presenting cells in the presence of PBMCs and multiple tool compounds. DP47, a non-pMHC-targeting TCB that only binds to CD3 (negative control), does not promote T cell activation and killing. Conversely, the nonspecific ESK1-like TCB (positive control) promotes CD8+ T cell expansion accompanied by dose-dependent T cell--mediated killing of multiple cell types, while WT1-TCB* recognizing the RMF-HLA-A2 complex with high specificity, leads solely to selective killing of WT1-expressing cells within kidney organoids under flow. Our 3D kidney organoid model offers a platform for preclinical testing of cancer immunotherapies and investigating tissue- immune system interactions. [ABSTRACT FROM AUTHOR]