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.