Abstract B063: Succinate based metabolic reprogramming of CAR-macrophages for ovarian cancer immunotherapy

Abstract Background: Despite the success of chimeric antigen receptor (CAR) T cell therapy in hematological malignancies, its efficacy in solid tumors remains limited. Macrophages, as key players in the innate immune response, offer unique advantages in the solid tumor context due to their ability to infiltrate tissues and phagocytose tumor cells. Preclinical studies have demonstrated that CAR-engineered macrophages (CAR-Ms) can penetrate solid tumors, initiate phagocytosis, and promote adaptive immunity. Early-phase clinical trials infusing CAR-Ms into patients have shown a favorable safety profile. However, within the immunosuppressive tumor microenvironment (TME), macrophages are often reprogrammed into tumor-promoting, M2-like tumor-associated macrophages (TAMs), which limit their therapeutic efficacy. Metabolic reprogramming is a critical driver of this polarization and TCA derived succinate has been shown to promote M1 phenotype in macrophages. In this study, we investigate whether succinate-loaded nanoparticles (PES NPs) can metabolically rewire CAR-Ms to maintain a pro-inflammatory (M1-like) phenotype and enhance antitumor activity in ovarian cancer (OC). Results: We synthesized polyethylene succinate (PES) nanoparticles (NPs) and analyzed their size, polydispersity, and zeta (ζ) potential using dynamic light scattering (DLS). The results showed a negative potential, indicative of stability, and suggested that succinate could be effectively encapsulated in nanoparticles. OC cells abundantly express mesothelin protein; therefore, mesothelin-targeting CARs were chosen to transfect mouse bone marrow-derived macrophages (BMDMs) or human monocyte-derived macrophages (HMDMs) to generate CAR-Ms. Our results showed that mouse and human CAR-Ms treated with PES NPs exhibited increased mitochondrial respiration and glycolysis, seen as elevated ECAR and OCR relative to vehicle control or cell-permeable dimethyl succinate ester (DMSA). Flow cytometry analysis revealed that PES treatment resulted in increased expression of markers of the classical M1 phenotype in both mouse and human CAR-Ms. The CAR-Ms also had elevated mRNA levels of proinflammatory cytokines like IL-6, TNFα, and IL1β, while anti-inflammatory cytokines like IL-10 remained low. Furthermore, IL-4-induced M2-like CAR-Ms could be reversed to an M1-like phenotype by PES NPs. Consistent with these results, PES NP-treated CAR-Ms exhibited increased phagocytic activity toward OC cells expressing mesothelin. Building on these in vitro results, the effect of PES NP-treated CAR-Ms on the reduction of tumor growth in OC tumor models will be further investigated. Conclusions: Our findings demonstrate that PES NPs effectively reprogram CAR-Ms to maintain a pro-inflammatory M1 phenotype. This reprogramming enhances their phagocytic activity and target cell killing in vitro. Collectively, these results support intracellular succinate delivery as a promising strategy to improve the efficacy of CAR-M-based therapies in OC. Citation Format: Remya Raja, Alexis Hand, Madhan Mohan Chandra Sekhar Jaggarapu, Jason Tonne, Richard Vile, Abhinav Acharya, Marion Curtis. Succinate based metabolic reprogramming of CAR-macrophages for ovarian cancer immunotherapy [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 B063.