Abstract A014: Autologous anti-5T4 CAR-T cell culture with live patient tumor slices reveals T cell intrinsic and microenvironmental suppression in pancreatic ductal adenocarcinoma
Authors: Evans Alice , Soyombo Mayowa , Hughes Daniel , Demetriou Constantinos , Moyce Laura , Russell Shannon , Willenbrock Frances , Blount Daniel , Ettorre Anna , Souberbielle Bernard , Mitrophanous Kyriacos , O'Neill Eric 
Published: February 2025

Abstract
Introduction: Culture of autologous patient CAR-T cells with organotypic tumor slices used to determine efficacy of a novel anti-5T4 CAR-T cell therapy in pancreatic ductal adenocarcinoma (PDAC). Methods: 5T4 expression was analyzed using human PDAC tumor tissue and healthy human pancreatic tissue. Immunohistochemistry (IHC) of fixed tissue and flow cytometry of disaggregated tumor samples were used to assess total and surface protein expression respectively. In vitro testing of anti-5T4 CAR-T efficacy was carried out using impedence-based cytotoxicity assays of healthy-donor derived CAR-T cells with PDAC cell lines. Tumor slices of 250μm were generated from punch biopsies of resected PDAC tumors and autologous CAR-T cells were generated prior to resection. CAR-T cells were cultured with live tumor slices and analyzed using live immunofluorescence (IF), fixed IF and IHC. Media was collected from slices for microenvironmental analysis using a cytokine array panel. CAR-T cells generated from both PDAC patient and healthy donor blood samples were analyzed using spectral flow cytometry. Results: Human PDAC tumors express high levels of 5T4 protein, and this expression correlates with poor survival in patients. 5T4 antigen is surface expressed in PDAC tumors and therefore accessible to CAR ligation. Anti-5T4 CAR-T cells kill PDAC cell lines efficiently in vitro, producing high levels of IL-2, IFN-y and Granzyme-B. Autologous patient CAR-T cells engage with target antigen in tumor slices within 1hr of co-culture. Co-localization with 5T4 is not seen in untransduced control (UTC) T cells. Modest calcium activation signals are seen upon autologous CAR-T cell contact with 5T4+ tumor cells. Despite co-localization and modest calcium activation there is no evidence of CAR-T cell killing in patient tumor slices from either cytokine production or cleaved caspase activation. Pre-treatment of CAR-T cells with tumor slice conditioned media causes a reduction in killing in vitro. Soluble factor multiplexed ELISA showed elevated levels of IL-6, IL-8, CCL2 and CXCL5 in tumour conditioned media. Healthy donor derived CAR-T cells kill more efficiently in vitro than patient-derived CAR-T cells. High dimensional flow cytometry of healthy donor and patient CAR-T cells will be conducted to investigate differences in phenotype. Conclusions: 5T4 is highly expressed in PDAC tumors and not in healthy control cells. Anti-5T4 CAR-T cells kill 5T4+ PDAC cell lines efficiently and selectively in vitro. Autologous 5T4 CAR T cells are unable to kill PDAC tumor cells ex vivo despite in vitro efficacy. CAR-T cells co-localize with 5T4+ tumor cells after 1hr of co-culture but fail to produce significant effector function. Multiple factors contribute to this lack of killing including local immune suppression and reduced patient T cell fitness. Organotypic tumor slices provide a clinically relevant model for testing autologous CAR-T cell therapy. Antigen distribution, native tumor architecture and tumor microenvironment can be studied across patients prior to clinical trial.
Citation Format: Alice Evans, Mayowa Soyombo, Daniel Hughes, Constantinos Demetriou, Laura Moyce, Shannon Russell, Frances Willenbrock, Daniel Blount, Anna Ettorre, Bernard Souberbielle, Kyriacos Mitrophanous, Eric O'Neill. Autologous anti-5T4 CAR-T cell culture with live patient tumor slices reveals T cell intrinsic and microenvironmental suppression in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A014.