Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma

Glioblastoma (GBM) is the most common primary malignant brain cancer, and is currently incurable. Chimeric antigen receptor (CAR) T cells have shown promise in GBM treatment. While we have shown that combinatorial targeting of 2 glioma antigens offsets antigen escape and enhances T-cell effector functions, the interpatient variability in surface antigen expression between patients hinders the clinical impact of targeting 2 antigen pairs. This study addresses targeting 3 antigens using a single CAR T-cell product for broader application. We analyzed the surface expression of 3 targetable glioma antigens (human epidermal growth factor receptor 2 [HER2], interleukin-13 receptor subunit alpha-2 [IL13R alpha 2], and ephrin-A2 [EphA2]) in 15 primary GBM samples. Accordingly, we created a trivalent T-cell product armed with 3 CAR molecules specific for these validated targets encoded by a single universal (U) tricistronic transgene (UCAR T cells). Our data showed that co-targeting HER2, IL13R alpha 2, and EphA2 could overcome interpatient variability by a tendency to capture nearly 100% of tumor cells in most tumors tested in this cohort. UCAR T cells made from GBM patients' blood uniformly expressed all 3 CAR molecules with distinct antigen specificity. UCAR T cells mediated robust immune synapses with tumor targets forming more polarized microtubule organizing centers and exhibited improved cytotoxicity and cytokine release over best monospecific and bispecific CAR T cells per patient tumor profile. Lastly, low doses of UCAR T cells controlled established autologous GBM patient derived xenografts (PDXs) and improved survival of treated animals. UCAR T cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.