Abstract Even though it is known that the immune response is highly relevant in ovarian cancer (OC), as demonstrated by the strong association between tumor infiltration by CD8 T cells and overall survival in high grade serous ovarian cancer, immunotherapy has not yet benefited patients with this disease. Therefore, there is a critical unmet need to identify new therapeutic targets and approaches that will improve outcomes for women with resistant disease. We used a novel culture system that uses a two-step activation process to elicit tumor-specific Type 1 (tumor-killing) T cells from the peripheral blood of OC patients. Briefly, the T cells are stimulated over the first two days with GM-CSF, MUC1 peptide antigens, and Toll-like receptor agonists (TLRa). After the second day in culture, the cells are washed and cultured in media containing IL-7 for the next 17 days. Using peripheral blood mononuclear cells (PBMCs) from 4 ovarian cancer patients, who had undergone between 1-3 prior lines of therapy, we have shown that all 4 were successfully activated and expanded with MUC1 antigens. Overall, the CD3+ cell population expanded from 5.7 – 12.6 fold over the 19 day culture, which was consistent with the sample from a healthy donor. Intracellular cytokine staining for IFNγ was used to determine MUC1-specific T cell responses at day 19. All OC patient T cells had increased reactivity to the MUC1-peptides as compared to the negative control peptide. Total MUC1-specificity ranged from 9.6 – 30.1% in CD4+ T cells and 6.6 – 16.8% in CD8+ T cells. We additionally performed high-parameter mass cytometry by time of flight (CyTOF) analysis using 34 antibodies targeting surface and intracellular markers. The FlowSOM algorithm followed by Marker Enrichment Modeling were used to identify major clusters of T cells and their associated phenotypes. Using this approach 12 major clusters of T cells were identified and overlayed on a tSNE plot. Of the 12 clusters, 7 represented CD8+ T cells and 5 represented CD4+ T cells. The day 19 clusters primarily represented T effector cells, T effector memory, and T central memory cells. The majority of day 19 cells expressed markers of effector function, including T-bet, EOMES, GRANZ-B, and GRANZ-K, which were present in both CD8+ and CD4+ populations. Exhaustion and regulatory markers were found to be low or absent in the day 19 cells. These results demonstrate that highly polyfunctional T cells with memory markers are generated from our innovative culture system using PBMCs from OC patients. These results provide preclinical evidence that will support future clinical trials of adoptive cell therapy using this culture system in OC. Citation Format: Brenda Ernst, Latha Pathangey, Michael Gustafson, Sandra Gendler, Marion Curtis. Rapid expansion of natural, MUC1-activated T cells from the peripheral blood of ovarian cancer patients for adoptive cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A086.