Enzymatic Remodelling of Tumour Microenvironment Enhances Anti‐CEACAM5 CAR T‐Cell Efficacy Against Colorectal Cancer

Chimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, anti-CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting primary CAR T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We found that CRC cells' glycocalyx is much thicker than that of the CAR T cell causing delayed activation. Oscillating calcium fluxes, indicative of non-sustained CAR T cell activation, are observed when CAR T cells interacted with CRC cells, which increased with increasing cell-seeding time. Significant reduction in cytotoxicity is observed on going from early to longer-seeded CRC monolayers. Imaging revealed that this effect correlated with a progressive loss of accessible CEACAM5 antigen on the CRC cell surface, possibly due to their sequestration in the intercellular junction, rendering CAR T cell engagement less effective. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T cell calcium fluxes. Similar enhanced interaction is observed after treatment of CRC cell monolayer with hyaluronidase, approved for use in humans. Enzymatic treatment significantly enhanced CAR T cell-mediated cytotoxicity and increased the percentage of TNF-α-secreting CAR T cells. We observed limited availability of CEACAM5 on human colorectal cancer tissues, whereas treatment with trypsin or hyaluronidase increased accessibility. Our results reveal why CAR T cells targeting CEACAM5 are ineffective and suggest possible routes to improved therapy for CRC.