P09.04 Ablation of prostaglandin E2-signaling through dual receptor knock-out in CAR T cells enhances therapeutic efficacy in solid tumors

Background Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment for hematologic malignancies but remains largely ineffective against solid tumors due to the immunosuppressive tumor microenvironment (TME). Prostaglandin E2 (PGE2) is a central mediator of tumor-driven immune suppression, acting as a local inhibitor of T cell function. We hypothesized that targeted ablation of PGE2 signaling could enhance CAR T cell activity in PGE2-rich TMEs by rendering them insensitive to PGE2-mediated inhibition. To test this, we disrupted PGE2 sensing via genetic deletion of its receptors EP2 and EP4.Material and Methods Using CRISPR/Cas9 gene-editing, we generated CAR T cells double deficient for EP2 and EP4 (EP2-/-EP4-/-). Next generation sequencing (NGS) of the CRISPR loci was performed to assess on-target editing efficiency as well as whole genome sequencing (WGS) of a representative donor to assess potential off-target editing. PGE2-signaling downstream of EP2 and EP4 (cAMP production and CREB phosphorylation), proliferation, activation, and cytotoxic capacity in the presence or absence of PGE2 were assessed in vitro. In vivo efficacy was evaluated in both syngeneic and human xenograft tumor models, including patient-derived xenografts from pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and neuroendocrine tumors (NET). T cell intratumoral accumulation and persistence were monitored by flow cytometric analyses.Results Successful generation of PGE2-insensitive EP2-/-EP4-/- CAR T cells was confirmed by NGS and showed complete abrogation of cAMP signaling and CREB phosphorylation upon PGE2 stimulation. No off-targets could be identified by WGS. While wild-type CAR T cells displayed impaired proliferation and reduced accumulation under PGE2 exposure, EP2-/-EP4-/- CAR T cells maintained robust proliferation and cytotoxicity. In vivo, loss of EP2 and EP4 markedly improved CAR T cell persistence and tumor infiltration, resulting in enhanced tumor control and prolonged survival across syngeneic and xenograft models. Notably, augmented anti-tumor activity was also observed when cocultured with patient-derived PDAC, CRC, and NET samples.DiscussionThese findings identify PGE2 as a potent barrier to CAR T cell efficacy in solid tumors and demonstrate that dual genetic disruption of EP2 and EP4 effectively shields therapeutic T cells from PGE2-mediated immunosuppression. Single receptor knockouts were insufficient to achieve this effect, underscoring the cooperative nature of EP2/EP4 signaling. Unlike systemic pharmacologic inhibition of PGE2 pathways, CAR T cell–intrinsic receptor ablation avoids perturbing physiological PGE2 functions. Targeting PGE2 signaling thus represents a compelling gene-editing strategy to enhance CAR T cell resilience and broaden their applicability in solid cancer therapy. S.K. has received honoraria from Plectonic, TCR2 Inc., Regeneron, Miltenyi, Galapagos, Cymab, Novartis, BMS and GSK. S.K. is an inventor of several patents in the field of immuno-oncology. S.K. and S.E. received license fees from TCR2 Inc and Carina Biotech. S.K. received research support from TCR2 Inc., Tabby Therapeutics, Catalym GmbH, Plectonic GmbH and Arcus Bioscience for work unrelated to the manuscript. R.W.J. is a member of the advisory board for and has a financial interest in Xsphera Biosciences Inc. R.W.J. has received honoraria from Incyte (invited speaker), G1 Therapeutics (advisory board), Bioxcel Therapeutics (invited speaker). R.W.J. has an ownership interest in U.S. patents US20200399573A9 and US20210363595A1. R.W.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict-of-interest policies. The remaining authors declare no competing interests.