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Background BAL0891 is a potent inhibitor of threonine tyrosine kinase (TTK, also known as MPS1) and polo-like kinase 1 (PLK1). 1 Inhibition of TTK and PLK1 induces chromosomal mis-segregation and micronuclei formation, activating the cGAS-STING pathway and type I interferon signaling, thereby enhancing antigen presentation and T-cell recruitment.2 This provides a rationale for combining BAL0891 with immune checkpoint inhibitors (ICIs).3 However, elevated concentrations of BAL0891 can paradoxically inhibit T-cell proliferation and function through mitotic disruption in activated lymphocytes. To precisely characterize and optimize these dual, concentration-dependent effects, we employed a biomimetic tumor microenvironment (TME) platform integrated with pharmacometric (PMx) modeling to translate findings into clinically relevant dosing strategies.Methods A vascularized three-dimensional TME platform incorporating triple-negative breast cancer patient-derived organoids, endothelial cells, fibroblasts, and peripheral blood mononuclear cells was utilized. BAL0891 was administered at multiple concentration levels. BAL0891’s direct tumor killing activity and immunomodulatory effects were assessed separately. Pharmacodynamic responses, including interferon-gamma (INF-γ) levels, tumor cell death, phospho-STING, and PD-L1 expression, were quantitatively assessed longitudinally over defined intervals. These data enabled the development of a concentration-response model featuring separate E max functions describing direct anti-tumor effects, immune activation (low-to-moderate concentrations), and T-cell suppression (higher concentrations). The concentration-response model was subsequently linked to a clinical pharmacokinetic (PK) model to predict anti-tumor efficacy across various clinically relevant combination regimens. PK parameters derived from the ongoing Phase I clinical trial (TTK-CS-101, NCT05768932) were integrated to simulate and identify optimal dosing regimens.Results BAL0891 demonstrated clear dose-dependent immune modulation. All assessed markers showed distinct longitudinal trends, justifying regimen optimization. BAL0891’s direct anti-tumor activity showed a clear dose-dependent increase. Additionally, maximal immune activation, indicated by peak INF-γ production and tumor cell killing, occurred at concentrations ranging from approximately 10 to 100 nM. Conversely, significant suppression of T-cell activity emerged at concentrations exceeding approximately >100 nM. PMx simulations indicated that optimal anti-tumor efficacy with an ICI could be achieved when the ICI was administered consistently 3-5 days after BAL0891 dosing, depending on the dose level.Conclusions By integrating advanced biomimetic and model-informed drug development technologies, our study successfully predicted optimal combination regimens for BAL0891 and an ICI. 3 4 This approach effectively demonstrated the critical importance of precise dose timing and optimization to maximize immunomodulatory and direct anti-tumor effects. Our findings highlight the broad applicability of this integrative strategy for optimizing similar immunotherapy combination regimens in clinical practice.Trial Registration NCT05768932References Lane HA, Zanini E, Forster-Gross N, et al. BAL0891: a novel, small molecule, dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) with potent single agent activity. Annals of Oncology 2022;33(Suppl 1):S18-S19. https://doi.org/10.1016/j.annonc.2022.01.051Kang M, Yoo S, Ma S. BAL0891 as a dual kinase inhibitor inducing anti-tumor immunity: A promising partner for immune checkpoint inhibitors. In: Proceedings of the AACR Annual Meeting 2025; Cancer Research 2025;85(8_Suppl_1):Abstract nr 2164. URL: https://aacrjournals.org/cancerres/article/85/8_Supplement_1/2164/760992Wang J, Kang M, Donnelly D, et al. Profiling patient response to TTK/PLK1 dual antagonist BAL0891 using 3D patient-derived organoid model in a microphysiological system. In: Proceedings of the AACR Annual Meeting 2025; Cancer Research 2025;85(8_Suppl_1):Abstract nr 3017. URL: https://aacrjournals.org/cancerres/article/85/8_Supplement_1/3017/756798Wang J, Kang M, Lee B, et al. TTK/PLK1 dual antagonist BAL0891 synergizes with pembrolizumab in a vascularized 3D tumor microenvironment model. In: Proceedings of the AACR Annual Meeting 2025; Cancer Research 2025;85(8_Suppl_1):Abstract nr 2169. URL: https://aacrjournals.org/cancerres/article/85/8_Supplement_1/2169/756917