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Background Immunotherapies have demonstrated significant clinical impact for cancers with high unmet medical needs. New generations of bispecific antibodies are entering the clinic for which upfront identification of patients with immunotherapy-sensitive cancer remains essential and challenging. Previously, we have demonstrated that our ex vivo 3D micro-tumor platform, which preserves the native tumor microenvironment (TME), is proven predictive for platinum-based therapy and correlates with clinical immune biomarkers. Here, we demonstrate that the platform also enables patient-specific drug sensitivity profiling and immune characterization for bispecific antibodies.Methods The presented study includes data from 14 tissue samples obtained from 12 ovarian cancer patients with primary, recurrent and progressive disease. Micro-tumors including native TME were isolated from fresh ascites (n=12) or pleural fluid (n=2), seeded and exposed to staphylococcus enterotoxin A (SEA) as positive control, single doses of pembrolizumab and dose ranges of chemotherapeutics and ubamatamab (CD3xMUC16 bispecific T cell engaging antibody in Phase 1/2 clinical trials). Drug response quantification was performed by extracting morphological features from high-throughput 3D imaging. Sensitivity was defined by statistically significant immune cell proliferation and micro-tumor killing. Supernatants collected after treatment were analyzed for secreted cytokines, chemokines and cytotoxic mediators using multiplex bead-based and proximity extension assay (Olink) immune biomarker profiling (n=5 patients). Immune cell populations were quantified by immunofluorescence staining.Results Treatment with ubamatamab resulted in a significant immune cell expansion in 43% (6/14) of ex vivo patient tissue samples. Among these, 2/6 (33%) also exhibited significant micro-tumor killing, representing complete functional responses. Ubamatamab responses were reproducible in subsequent samples from the same patients, demonstrating assay consistency. Baseline cytokine profiling revealed substantial patient heterogeneity. Patients with low baseline cytokine levels showed ex vivo resistance to ubamatamab. Notably, responders with effective micro-tumor killing exhibited robust and simultaneous upregulation of effector T cell activation biomarkers (IFN-γ, granzyme B, perforin, and Fas ligand) following treatment. Importantly, ex vivo responses to ubamatamab did not correlate with sensitivity to pembrolizumab or chemotherapy, suggesting this therapy may benefit a distinct patient population.Conclusions This study reports the classification and characterization of patient-specific sensitivity to ubamatamab, a bispecific antibody in clinical development for ovarian cancer. Our ex vivo 3D micro-tumor platform enables quantification of immune responses and identification of predictive biomarkers that will facilitate patient stratification for novel immunotherapies.Ethics Approval Ovarian cancer patients from the Netherlands were enrolled under a Western IRB-approved protocol into the TUMOVCA study (protocol number IRB P18.032). The IRB (P18.032) reviewed the protocol and confirmed the trial not being subject to the WMO, as patients received standard-of-care treatment and were not going through extra invasive procedures.