Abstract Rationale: Most ovarian tumors show some level of infiltration by T lymphocytes, and increasing levels of tumor-infiltrating lymphocytes (TIL) correlate with improved outcomes. The field of cancer immunotherapy has rapidly evolved and made a significant impact on many tumor types, including gynecologic cancers. However, single agent immune therapies have not yet demonstrated significant clinical success in epithelial ovarian cancer (EOC). We hypothesize that a combination of intraperitoneal (IP) chemotherapy (cisplatin) with dual agent immunotherapy using intravenous (IV) pembrolizumab (anti-PD1) and IP rintatolimod (TLR-3 agonist) will help overcome immune suppressive mechanisms for improved tumor response by promoting increased T cell chemotaxis and cytolytic function. Methods: To test our hypothesis, we are running an investigator initiated, phase II, single arm, efficacy/safety trial (NCT03734692). Peritoneal fluid aspiration (IP wash) was performed in 13 patients at multiple time points during each cycle of treatment. Washes were collected on Days 1-3 of each cycle, before and after each treatment. Washes from cycle 1, 4 and 6, were included in this analysis. The MesoScale Delivery (MSD) platform was used to profile different biomarkers in the peritoneal samples throughout treatment. Results: In this ongoing Phase II study, a total of 17 patients were enrolled and 13 were evaluable for response. The observed clinical responses were: 2 complete responses (15.4%), 3 partial responses (23.1%), 3 stable disease (23.1%), 5 progressions (38.4%) for an clinical benefit rate (CR+PR+SD) of 61.6%. From 13 patients, 7 IP wash samples were collected at serial time points. MSD measurements in IP washes revealed an acute increase in granzyme B, perforin, TNF alpha, CXCL9, CXCL10, CXCL11 after treatment (p<0.05). Longitudinal data revealed a progressive increase in some biomarkers in the locoregional environment; CXCL9, CXCL10, CXCL11, perforin and TNF alpha were all increased from baseline levels at cycle 1 to baseline of cycle 6 (p<0.05). CXCL12 was also increased acutely after treatment (p<0.05). Discussion: This is a novel, triple drug combination for EOC that leads to a locoregional acute response with an increase in biomarkers associated with T cell chemotaxis and cytolytic function. Longitudinal comparison of these biomarkers showed a gradual, durable response over time in T lymphotactic CXCR3 ligands. While an acute response was also noted in CXCL12, a marker of secondary suppression, a progressive increase over time was not detected. While these are interim analyses and ongoing evaluation of additional samples from the trial are needed to confirm our findings, the preliminary results show promise for novel treatment regimen. Citation Format: Mackenzy Radolec, Brian Orr, Mary Strange, Lixin Zhang, Haider Mahdi, Sarah Taylor, Gregory Willis, Robert Edwards, Anda Vlad. Combined loco-regional and systemic, triple agent chemoimmunotherapy increases biomarkers of T cell chemotaxis in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6134.