ANALYSIS OF IL13Rα2-CAR T CELLS IN MOUSE MODEL OF GLIOBLASTOMA

The outstanding efficacy of T cells modified to express chimeric antigen receptors (CAR) for hematological malignancies promises hope that they can be programmed to target and kill solid tumors. As the current standard of care for glioblastoma (GBM) only extends patient survival minimally, CARs can provide an exciting new option for the treatment of this incurable disease. We have generated a CAR expressing the specific single chain antibody (scFv47) against interleukin-13 receptor α 2, (IL13Rα2), a tumor associated antigen expressed highly by glioma tissues. In our recent studies, we have demonstrated that scFv47 CAR T cells are efficacious in killing human glioma cells in vitro and in vivo in athymic mice. Understanding how these CAR T cells behave in the GBM microenvironment and interact with the host immune system is critical for the development of successful CAR T cell therapies. Therefore, in our studies, we generated and compared the efficacy of mouse CAR T cells equipped with two different linkers, short (SL, mouse IgG hinge domain) and long (LL, extracellular domain of murine CD28), connecting scFv47 with transmembrane domains and signaling domains of CD28 and CD3ζ. Interestingly, the linker type was critical to the efficacy of the CAR, as the in vitro 51Cr release/killing assay showed that SL-CD28ζ CAR T cells were more effective in lysing GBM cells. This further translated to the in vivo studies, where SL- S CD28ζ CAR T cells significantly extended the survival of mice bearing syngeneic GL261 or SMA560 gliomas modified to express human IL13Rα2. Multicolor flow analysis of tumor tissues showed that SL-CD28ζ CAR T cells persisted at three and seven days after intracranial injection into established GBMs. Interestingly, we observed a reduction of host Tregs at the three day time point, with minimal contribution of CARs to the overall CD4 compartment in the tumor. There was a significantly increased presence of host CD8α+ DC in the brain in response to SL-CD28ζ CAR and not controls suggesting that CARs can enhance recruitment of functional DCs to the tumor. Furthermore, there was a reduced presence of granulocytic myeloid-derived suppressor cells (MDSCs) in the brain and spleen of SL-CD28ζ CAR treated mice. The systemic reduction of MDSCs by functional CARs suggests they are influencing the chemotactic signals used by the tumor to recruit immunosuppressive cells. In conjunction with our data of IL13Rα2-CAR T cell immunotherapy in an immune-compromised model, our study of IL13Rα2-CAR T cells in an immune-competent model shows great promise for the improvement of GBM immunotherapy. We showed that IL13Rα2-CAR T cells are efficacious in killing GBM both in vitro and in vivo. Furthermore, for the first time, we showed that CAR T cell therapy positively modulates the immune landscape of the glioma bearing mice, likely contributing to the antitumor activity of CAR T cells.