Abstract PR006: Metabolic reprogramming of ovarian CD137+ TILs for treatment

Abstract Background: Ovarian cancer remains the most lethal gynecologic malignancy and a leading cause of cancer-related deaths among women. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) has shown success in melanoma and correlates with survival in ovarian cancer. However, its application remains limited, partly due to inefficient expansion of functional, tumor-specific T cells. Methods: We developed a novel ex vivo expansion platform for ovarian TILs, integrating metabolic and phenotypic reprogramming with selective enrichment for tumor-reactive populations. Tissue from nine patients with stage III/IV high-grade epithelial ovarian carcinoma was collected for banking, NGS, and TIL expansion. Four had matched TILs expanded pre- and post-neoadjuvant chemotherapy. The initial priming phase employed our published cytokine cocktail designed to induce Th1/17 and Th17/Tc17 polarization, to promote effector differentiation while preserving stemness. TILs were cultured for five days in this cocktail to enhance metabolic fitness and anti-tumor potential. CD137+ (4-1BB+) T cells were isolated based on activation-induced co-stimulatory expression and subjected to rapid expansion using a G-Rex-based bioreactor system with irradiated feeder cells, anti-CD3, and low-dose IL-2. Expanded TILs were characterized by multicolor flow cytometry, CD107a degranulation, IFN-γ ELISA, and single-cell RNA sequencing (scRNA-seq), and benchmarked against TILs expanded using the conventional protocol from NCI. Results: Our approach reliably produced 15–20 billion viable TILs per patient within 30 days, achieving >95% viability and ∼2500-fold expansion. CD137+ enrichment enhanced tumor reactivity, as shown by increased IFN-γ secretion and degranulation with autologous tumor and high-grade serous ovarian cancer cell lines. scRNA-seq revealed enriched cytotoxic CD8+ T cells and mucosal-associated invariant T (MAIT) cells, with reduced FOXP3+ regulatory and non-cytotoxic CD4+ subsets, compared to TILs from matched blood, tumor, and those expanded with high-dose IL-2 in the NCI protocol. CD8+ proliferative and cytotoxic T cell subsets were enriched under the hybrid CD137-sorted protocol. Transcriptomic profiling showed that CD8+ proliferating T cells from the hybrid group downregulated key metabolic genes, including SLC7A5 and LDHB, suggesting a rewired metabolic state that may support persistence. Similarly, CD8+ cytotoxic T cells showed chromatin remodeling, with upregulation of histone demethylases KDM5A and KDM7A, and downregulation of CHD7 and JARID2, indicating transcriptional plasticity. MAIT cells also showed increased activation markers (IFNG, CCL20). Expanded TILs exhibited reduced expression of exhaustion markers PD-1 and CD38 at the protein level. Conclusion: Metabolic reprogramming combined with CD137-based enrichment enables robust expansion of metabolically reprogrammed, tumor-specific TILs from ovarian tumors. This strategy offers a promising foundation for advancing TIL-based ACT in ovarian cancer. Citation Format: Paramita Chakraborty, Stephanie Mills, Jadan Zavestoski, Satyajit Das, Suganya Subramanian, Nicolina Mascia, Jian Liu, Stefano Berto, Brian Orr, Shikhar Mehrotra, Stephanie Mills. Metabolic reprogramming of ovarian CD137+ TILs for treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr PR006.