Cytokine Programming Combined with 4-1BB Enrichment Enhances Ovarian TIL Expansion and Function 2260346

Abstract Introduction Ovarian cancer remains the most lethal gynecologic malignancy and the fifth leading cause of cancer-related deaths in women worldwide. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) has shown durable efficacy in melanoma. In ovarian cancer, higher TIL levels correlate with improved survival; however, clinical translation of TIL-based ACT remains limited by challenges in expanding functional, tumor-specific T cells ex vivo. Methods Tumor tissue from 12 patients with stage III/IV high-grade epithelial ovarian carcinoma was used for TIL expansion, including matched pre- and post-chemotherapy samples. During priming, a cytokine cocktail promoted Th1/17 and Tc1/Tc17 polarization, enhancing effector differentiation while maintaining stemness. After five days, CD137+ (4-1BB+) T cells were enriched and expanded in G-Rex bioreactors with irradiated feeders, anti-CD3, and low-dose IL-2. TILs were evaluated by flow cytometry, CD107a degranulation, IFN-γ ELISA, and scRNA-TCR-seq, compared to the NCI high-dose IL-2 protocol. Results Our method generated 15-20 billion TILs within 30 days, with >95% viability and ∼2,500-fold expansion. 4-1BB+ enrichment reduced expression of exhaustion markers PD-1 and CD38 and enhanced tumor reactivity, marked by increased IFN-γ secretion with autologous tumor and ovarian cancer lines. scRNA-seq revealed more cytotoxic CD8+ and fewer CD4+ Th2 cells compared to the NCI protocol. Transcriptomic profiling also showed CD8+ T cells with reduced metabolic activity and altered chromatin remodeling, consistent with persistence and transcriptional plasticity. TCR sequencing indicated that bulk tumor and NCI-expanded TILs were diverse with many singletons, while 4-1BB+ cells were oligoclonal and dominated by hyperexpanded CD8+ effectors. Conclusion Cytokine programming combined with 4-1BB -based enrichment enables robust expansion of metabolically reprogrammed, tumor-specific TILs, presenting a promising ACT strategy for ovarian cancer. Funding Source NIH-HL-007260R01 CA250458, R01 CA236379, GREX Grant Program Topic Categories Translational and Interventional Immunology (TI)