Ex vivo evaluation of a MUC1-targeted bispecific T-cell engager in ovarian tumoroids using Nilogen's 3D-EXpress platform

BACKGROUND: Mucin 1 (MUC1) is a relevant target for bispecific T-cell engagers due to its expression in epithelial malignancies. Nilogen Oncosystems' 3D-EXpress ex vivo tumoroid platform enables direct assessment of patient-specific responses to immunotherapies in a physiologically preserved tumor-immune microenvironment. This study evaluated the activity of a commercially available recombinant Anti-CD3 x Anti-MUC1 bispecific T cell engager antibody (MUC1-TCE) in cryopreserved ovarian tumor-derived tumoroids to characterize immune activation using Nilogen's integrated platform combining flow cytometry, cytokine profiling, and RNA transcriptomic analysis. METHODS: Pathology reports were reviewed for all tissues and confirmed characteristic histological features consistent with high-grade ovarian carcinoma. Tissues were evaluated for MUC1 expression, showing high to low levels. Tumoroids were generated by mechanical dissociation without enzymatic digestion or in vitro propagation. After thawing, tumoroids were treated with MUC1-TCE, isotype control, or positive control. Multiparametric flow cytometry measured T-cell activation. Parallel supernatants were collected for multiplex cytokine profiling, and RNA was isolated for downstream transcriptional analysis. RESULTS: MUC1-TCE treatment induced clear immune activation across multiple tissues. CD4+ T cells upregulated 4-1BB, CD25, CD69, OX40, and PD-1, while CD8+ T cells upregulated 4-1BB, CD25, ICOS, and OX40. Cytokine profiling showed strong induction of inflammatory mediators, with IFN-gamma and TNF displaying the greatest response. Preliminary RNA analysis revealed enrichment of immune-activated, inflammatory, and cytokine-driven pathways consistent with effector engagement. CONCLUSIONS: These findings demonstrate the feasibility of using cryopreserved patient-derived tumoroids for ex vivo testing of bispecific T-cell engagers while preserving the native tumor-immune microenvironment. Integration of cytokine measurements, transcriptomic readouts, and immune phenotyping provides a scalable approach for characterizing mechanisms of action of MUC1-targeted agents and supports continued development of bispecific immunotherapies for solid tumors.