10.01 Prostanoid-insensitive chimeric antigen receptor modified T cells mediate therapeutic efficacy in solid cancer models

Background Chimeric antigen receptor (CAR) T cell therapy has transformed the landscape of hematologic malignancy treatment. However, its successful translation to solid cancer treatment remains challenging, primarily due to the immunosuppressive tumor microenvironment (TME). Prostaglandin E₂ (PGE₂) has emerged as a pivotal player in TME immunosuppression, whereby its impact on T cell function has been recognized as a novel immune checkpoint. This study focuses on enhancing CAR T cell efficacy against solid tumors by disrupting PGE₂ signaling through the targeted knockout of its receptors, EP2 and EP4. Materials and Methods Utilizing CRISPR/Cas9 technology, we generated double knockout CAR T cells deficient in EP2 and EP4. In vitro analyses assessed the impact of PGE₂ on CAR T cell functions, including proliferation, activation, and cytotoxicity. To translate these findings to a human system, we conducted tumor growth and survival experiments using a xenograft mouse model. Additionally, T cell tracking was performed to elucidate the fate of EP2-/-EP4-/- CAR T cells in vivo. Results We could successfully generate murine as well as human EP2 and EP4 single and double knockout CAR T cells, which showed complete abrogation of PGE₂ signaling, as indicated by suppressed cAMP production and CREB phosphorylation. While wild type CAR T cells were suppressed in their proliferative abilities by PGE₂, their capacity to become activated remained unaffected. PGE₂-induced reduction in CAR T cell numbers ultimately compromised their anti-tumor activity both in vitro and in vivo. This effect was rescued by the EP2 and EP4 double knockout, leading to increased EP2-/-EP4-/- T cell accumulation within tumors. As a consequence, bettered therapeutic efficacy and prolonged survival were achieved in a xenograft mouse model. Conclusions Our findings underscore the detrimental impact of PGE₂-mediated suppression on CAR T cell efficacy in the TME, highlighting the potential to rescue therapeutic cells through EP2 and EP4 knockout. Notably, single knockouts of either receptor proved insufficient to shield CAR T cells from PGE₂. Genetic ablation of EP2 and EP4 presents a promising strategy to shield CAR T cells from PGE₂ without disrupting healthy PGE₂ signaling and encourages further exploration and development of this novel therapeutic strategy. J. Dörr: None. L. Gregor: None. S.B. Lacher: None. A. Öner: None. S. Lesch: None. S. Michaelides: None. L. Majed: None. L. Fertig: None. E. Carlini: None. D. Andreu Sanz: None. D. Briukhovetska: None. S. Stock: None. A. Gottschlich: None. J.P. Böttcher: None. S. Kobold: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; TCR2 Inc, Catalym, Plectonic, Arcus Biosciences, Tabby Therapeutics. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; GSK, BMS, Novartis, Miltenyi Biotherapeutics, TCR2 Inc. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TCR2 Inc, Carina Biotech.