ABSTRACT: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.
