Background This study explores the synergistic potential of combining mouse mesothelin-specific chimeric antigen receptor (mmeso-CAR) T cells and a CD40 agonist (αCD40) to enhance CAR T cell and overall immune response against pancreatic ductal adenocarcinoma (PDAC) in syngeneic mouse models.
Methods The subcutaneous syngeneic PDAC model was established using a KPC-derived cell line. Mice were treated with mmeso-CAR T cells, αCD40, or combinations of both. In vivo therapeutic efficacy was evaluated in endpoint and time course models, monitoring tumor volume and histological changes over time, and assessing CAR T cell and immune cell distribution and activation in secondary lymphoid organs (SLOs): tumor draining lymph node (TdLN) and spleen, and the tumor microenvironment (TME). Our methodology encompassed real-time live cell assays, in vivo imaging, multiplex cytokine assays, multi-parametric flow cytometry, histology, RNAscope, digital spatial profiling, and scRNAseq. Additionally, the combination treatment is currently being evaluated in an orthotopic syngeneic model of triple-negative breast cancer (TNBC).
Results Combining mmeso-CAR T cells with αCD40 yielded improved tumor control and long-term survival outcomes. αCD40 treatment induced significant tumor necrosis within 24 hours (39.5 ± 29.1% and 33.8 ± 20.5% of tumor area, with or without mmeso-CAR T cells, respectively). The necrotic effect persisted after seven days when combined with mmeso-CAR T cells (25.9 ± 20.7% versus 2.1 ± 3.3% with αCD40 alone), associated with a greater reduction in tumor weights and PanCK/mesothelin+ tumor areas (figure 1). αCD40 treatment promoted the expansion of mmeso-CAR T cells and modulated their activation in SLOs and within the TME. The combination therapy engaged APCs, host T and NK cells, increasing recruitment and activation in SLOs (figure 2) and in the TME, associated with elevated blood levels of pro-inflammatory cytokines and chemokines. ScRNAseq analysis on immune cells from tumor and TdLN confirmed the involvement of both CAR-dependent and independent antitumor responses.
Conclusions This study unveils the synergistic effect of combining mmeso-CAR T cells and αCD40 in delaying tumor growth in a syngeneic PDAC model. The combination therapy led to rapid and sustained tumor necrosis with increased infiltration and activation of immune cells in the SLOs and the TME. Our comprehensive characterization provided valuable mechanistic insights into the underlying synergistic mechanisms. Ongoing evaluation of the mmeso-CAR T cells/αCD40 therapy in a syngeneic model of TNBC aims to assess the effectiveness in various solid tumors expressing mesothelin. These findings may open potential applications of meso-CAR T cells/αCD40 combination therapy in the clinic.
Acknowledgements We express gratitude to the following facilities at the University of Pennsylvania: the Stem Cell and Xenograft Core (SCXC) for providing equipment for in vivo procedures; the Pathology Core at the Children’s Hospital of Philadelphia for tissue processing, immunohistochemistry and image acquisition; the Comparative Pathology Core at the School of Veterinary Medicine for immunohistochemistry, image acquisition, and pathological assessment; and the Translational and Correlatives Studies Laboratory at the Center for Cellular Immunotherapies for support and access to the nanoString® GeoMx platform. We thank the Beatty, Stanger and Tchou Laboratories at the Center for Cellular Immunotherapies for providing PDAC and TNBC cell lines.
Ethics Approval The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved animal experiments (protocol n°804226). Animal procedures were performed in the animal facility at the University of Pennsylvania in accordance with Federal and Institutional IACUC requirements.
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