Targeting the extracellular matrix to improve CAR-T cell therapy against Prostate Cancer

Prostate cancer remains a major cause of cancer-related death among men, particularly in cases of metastatic or treatment-resistant disease. While conventional therapies have shown efficacy in managing localized prostate cancer, their impact on advanced stages remains limited. Adoptive cell therapy (ACT) with T lymphocytes expressing Chimeric Antigen Receptors (CAR-T) has emerged as a promising immunotherapy to treat hematological malignancies. However, CAR-T cell therapy application in solid tumors remains challenging, mainly because the immunosuppressive tumor microenvironment (TME) limits proper T cell functionality. In particular, the tumor extracellular matrix (ECM), which is rich in collagen and hyaluronic acid (HA), blocks the infiltration of immune cells. It is therefore needed to endow CAR T cells with tools that will allow T cells to overcome the ECM barrier and TME immunosuppression. In this project, we present a promising strategy to enhance CAR-T cells' therapeutic efficacy in those solid tumors that are characterized by a rich and stiff ECM and are resistant to common therapies, such as prostate cancer (PCa), by inducing the co-expression of ECM-degrading enzymes. Specifically, since the PCa ECM is rich in type I collagen, HA and fibronectin, we selected enzymes sensitive to these substrates such as collagenases and hyaluronidases. We selected either a soluble or membrane-bound form of the enzymes to investigate whether they might improve differently CAR-T cell infiltration. The PSMA CAR-T cells co-expressing the ECM-degrading enzyme (PSMA CAR.E) were endowed with high in vitro invasion capacity, thanks to the efficient expression of the ECM-degrading enzymes. Specifically, the co-expression of the transmembrane mMMP14 and the soluble sMMP8 collagenases showed a significantly improved infiltration of the PSMA CAR.E T cells in collagen and fibronectin-rich matrix. Interestingly, we did not observe any beneficial role in the expression of the soluble metalloprotease compared to the transmembrane one. The infiltration of PSMA CAR.E T cells co-expressing either membrane-bound hyaluronidase mPH20 or the soluble form sPH20 was assessed with an HA-based matrix, detecting a significantly enhanced infiltration of the engineered T cells, compared to PSMA CAR T cells. Remarkably, we detected a more pronounced infiltration of the PSMA CAR.sPH20 T cells compared to the PSMA CAR.mPH20 T cells. Killing assays have also highlighted that PSMA CAR.E T cells have strong antigen-specific cytotoxic potential compared to untransduced primary T cells. Moreover, PSMA CAR.E T cells are characterized by a memory-like phenotype, which is reported to be a favorable factor for the application of CAR-T cell products in in vivo settings. In this study, we observed that the expression of an ECM-degrading enzyme endowed the CAR-T cells with an improved invasion capacity, which represents a promising strategy to enhance their therapeutic efficacy in solid tumors. Another major challenge for a successful CAR-T cell application in prostate cancer is related to the on-target off-tumor activation of the engineered T cells, which may result in unintended damage to healthy tissues and potentially severe toxicities. Recently, small-molecule responsive "ON-switch" CARs dependent on caffeine have been developed, allowing precise control over T-cell activation while maintaining antigen specificity. We applied the caffeine-dependent ON-Switch system to the PSMA CAR molecule (CaffPSMA CAR), assessing CaffPSMA CAR T cell-specific activation upon caffeine administration only when co-cultured with PSMA-expressing prostate cancer cells. Furthermore, we demonstrate that changes in a co-stimulatory domain do not affect the caffeine-dependent dimerization and give no aspecific CAR signalling activation. To conclude, we overall demonstrated that the caffeine-regulated molecular switch system can be successfully applied to the PSMA CAR in human primary T cells