Generation and characterization of murine CAR-T cells to investigate the impact of immunotherapy in pancreatic adenocarcinoma

Cancer is one of the leading causes of mortality worldwide, also due to the limitations of existing therapies in managing treatment-resistant forms. Immunotherapies have emerged as a revolutionary approach to fight cancer by harnessing the body’s immune system to specifically target and eliminate malignant cells. In particular, adoptive cell therapy (ACT) employing chimeric antigen receptor- (CAR) engineered T-cells has shown remarkable success in treating hematological malignancies. CAR-T treatment of solid tumors has, however, faced significant obstacles, primarily due to the inherent immunosuppressive nature of the tumor microenvironment (TME) and the physical barrier represented by the dense stroma. Indeed, the efficacy of CAR-T cells in solid tumors is significantly hindered by the poor infiltration of the immune cells through the collagen- and hyaluronic acid- (HA) rich extracellular matrix (ECM). Thus, implementing CAR-T cells is crucial to overcome these hurdles. In this project, we present a promising strategy to enhance CAR-T cells infiltration in solid tumors characterized by a stiff ECM, such as pancreatic ductal adenocarcinoma (PDAC). We aim to co-engineer MSLN-targeting CAR-T cells with ECM-degrading enzymes, namely the collagenase MMP14 and the hyaluronidase PH20. With the perspective of evaluating CAR.enzyme-(CAR.E)T cells penetration and interactions within the TME in immunocompetent, syngeneic PDAC mouse model that more closely recapitulates human cancer, we developed this strategy in a fully murine setting. Here, we present the optimization of a protocol to generate murine MSLN CAR.E T-cells, mainly focusing on improving the efficiency of retroviral vector production, as well as the activation and transduction of the murine T-lymphocytes. These advancements resulted in improved murine T-lymphocytes viability and robust expansion, along with the generation of a high proportion of CAR-positive T-cells which show a sustained CAR expression and an efficient enzyme production. Furthermore, the engineered T-cells exhibited potent in vitro cytotoxic activity against mMSLN-positive PDAC cell lines, proving their ability to fulfill their designated purpose. Overall, this work demonstrates the successful generation of murine MSLN CAR.E T-cells, presenting a promising strategy to enhance therapeutic infiltration and efficacy in solid tumors embedded in a stiff ECM, such as PDAC, and to study the therapy’s effects on the TME, paving the way for the identification of novel potential therapeutic targets.