Endogenous bystander killing mechanisms enhance the activity of novel FAP-CAR-T cells against glioblastoma

Abstract

AbstractCAR-T cell therapies are being intensely investigated as a novel immunotherapy approach for glioblastoma (GBM), but so far clinical success has been limited. We recently described FAP as an ideal target antigen for GBM immunotherapy, with expression on tumor cells and tumor blood vessels occurring frequently in patients’ tumors but with very limited expression in healthy tissues. Here, we generated a novel FAP-targeting CAR with CD3ζ and CD28 signaling domains and tested the resulting CAR-T cells for their ability to lyse GBM cells in vitro and in vivo. FAP-CAR-T cells showed target specificity against model cell lines and exhibited potent cytotoxicity against patient-derived glioma neural stem (GNS) cells. Remarkably, complete destruction of tumor cells was observed even where the antigen was expressed by a minor subpopulation of cells, indicating a bystander killing mechanism. Using co-culture assays, we confirmed the ability of FAP-CAR-T cells to mediate bystander killing of antigen-negative tumor cells, but only after activation by antigen-expressing target cells. This bystander killing effect was at least partially mediated by soluble factors. We also observed that the non-transduced fraction of the CAR-T cell product could be activated via T cell-secreted IL-2 to mediate antigen-non-specific killing, further amplifying the bystander effect. FAP-CAR-T cells controlled without overt toxicity the growth of subcutaneous tumors created using a mixture of antigen-negative and antigen-positive GBM cells. Together, our findings advance FAP as a leading candidate for clinical CAR-T cell therapy of GBM and highlight under-recognized antigen non-specific mechanisms that may contribute meaningfully to the antitumor activity of CAR-T cells.