OX40–heparan sulfate binding facilitates CAR T cell penetration into solid tumors in mice

Although chimeric antigen receptor (CAR)-modified T cells have shown great success in treating B cell malignancies, they have demonstrated only limited efficacy against solid tumors. Here, we designed a CAR by integrating an antigen-independent OX40 that showed superior antitumor efficacy against multiple solid tumors. We unexpectedly found, through a CRISPR-Cas9-based whole-genome screen, that heparan sulfate is a ligand for OX40. We found that heparan sulfate can directly bind OX40 at the biochemical and cellular levels and that the interaction of heparan sulfate and OX40 activated the AKT, MAPK, and NF-κB signaling pathways. Functionally, the heparan sulfate-OX40 interaction enhanced cell adhesion and CAR T cell functional binding avidity to tumor cells. In vivo, OX40-expressing CAR T cells exhibited increased solid tumor infiltration and persistence dependent on the OX40-heparan sulfate interaction. Our findings provide insights into a glycan-costimulation interaction that is capable of regulating T cell immunity and has potential application in CAR T cell optimization.