Abstract Introduction: Clinical trials of mesothelin (MSLN) chimeric antigen receptor (CAR) T-cell therapy have shown limited efficacy in solid tumors like ovarian cancer (OVCA), emphasizing the need to enhance CAR T cell performance. This study evaluated whether combining MSLN-CAR T cells with drugs, either sequentially or concurrently, could achieve synergistic anti-tumor effects. Methods :MSLN-directed CAR T cells were generated using two constructs with CD28 (M28z) or 4-1BB (MBBz) co-stimulatory domains. A high-throughput drug screen of 528 compounds was conducted on MSLN-expressing OVCA cells in combination with CAR T cells, assessing three conditions: (1) simultaneous drug and CAR T exposure, (2) short drug pre-treatment of OVCA, and (3) extended drug pre-treatment before CAR T cell introduction. Drug-induced modulation of CAR T cell cytotoxicity was assessed by comparing CAR T cell-mediated killing, drug-only effects, and drug-CAR T cell combination effects on OVCA cells. Results: Synergy between drugs and CAR T cells depended on treatment timing and co-stimulatory domains. Chemotherapeutics enhanced CAR T cell cytotoxicity when administered as a pre-treatment, while epigenetic modulators and kinase inhibitors were more effective with concurrent CAR T cell application. Cisplatin and cytarabine significantly boosted MBBz CAR T activity, while idarubicin enhanced M28z CAR T cells. Certain agents, like vinflunine, gemcitabine, and the PARP inhibitor rucaparib, enhanced efficacy across both constructs. Donor-specific responses revealed two distinct donor clusters: one showed synergy with chemotherapeutics, and the other with targeted agents Conclusions: Combining drugs with CAR T cell therapy could improve the treatment efficacy in ovarian cancer. Pre-treatment with specific chemotherapeutics enhanced CAR T cell cytotoxicity, while simultaneous treatment with certain targeted therapies was also effective. The effects of CAR construct type and inter-donor variability highlight the potential for precision medicine approaches. These findings support further exploration of personalized therapeutic combinations to optimize CAR T cell efficacy in ovarian cancer and improve patient outcomes. Citation Format: Lucía Rico Pizarro, Thomas Poiret, Greta Gudoitytė, Olli Kallioniemi, Brinton Seashore-Ludlow, Isabelle Magalhaes, Tom Erkers. Enhanced efficacy of anti-mesothelin CAR T cells in ovarian cancer through drug combinations: Impact of treatment kinetics, co-stimulatory domain, and donor variability [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A017.
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