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Chimeric antigen receptor (CAR)-T cells have had a significant effect in hematological diseases, with a recent study confirming that the cells can last for ten years in vivo. Unfortunately, to date, CAR-T cells have had little effect in solid tumors. One cause of the treatment difficulty is antigenic heterogeneity. Our experience with CAR-T cells in glioblastoma suggests that addressing antigenic heterogeneity may improve the clinical efficacy. Targeting the epidermal growth factor receptor (EGFR) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) individually could lead to tumor control in the short term, but it will be marked by tumor recurrence through antigen loss or downregulation in the long term. This resistance mechanism can be inhibited by combinatorial targeting. To this end, we have designed a bicistronic construct that targets both EGFR and IL13Rα2 simultaneously, to decrease the potential of antigen escape and tumor recurrence. The bicistronic CAR-T cells employ an “OR” logic gate to modify the T cells and kill the tumor cells in vitro and in vivo, in order to retain effectiveness when one target antigen is lost but the second is still present. Our experimental results demonstrate that and validate the safety and possibility of clinical translation. In addition, the efficiency of transfecting T cells with the single bicistronic vector was higher than two single vectors on monovalent CARs. In summary, the bicistronic CAR-T structure is a promising strategy to address the clinical problem of targeting antigenic heterogeneity in solid tumors and potentially level the barriers for targeted T-cell therapy. Educational Objectives: After completion, participants will be able to explain the importance of antigenic heterogeneity in solid tumors in the context of tumor escape. Participants will also be able to identify a strategy to reducing the problem of antigen escape and tumor recurrence through the implementation of bicistronic CARs.

Identifying factors that contribute to the aggressiveness of glioblastoma (GBM) is crucial for treating this deadly brain tumor. We demonstrate here that CD70, a member of the TNF family, is overexpressed by tumor cells in a subset of patients with low grade glioma and GBM. The elevated gene and protein expression is associated with increased tumor grade and recurrences. CD70 expression on primary GBM is correlated with T cells infiltration and tumor MHC class I expression which could potentially promote antitumor immunity based on our result that T cell infiltration is negatively associated with GBM proliferation. However, no correlation between T cell infiltration and prolonged survival is seen, suggesting that other factors may exist to suppress T cell function. Analysis from 155 primary GBM patients using RNA-seq data culled from TCGA indicates that the gene expression of CD70 is highly correlated with markers (CD4, CD25, CD163 and CD14) commonly expressed on immune suppressive cells such as Tregs and tumor associate macrophages. This result is in line with additional finding that CD70 positive tumors display a predominantly mesenchymal gene expression signature, a subtype of GBM that has greater infiltration by immune cells than other subtypes. In addition, tumors derived from patients with MGMT promoter un-methylation express high levels of CD70. Furthermore, our results suggest that CD70 is a key driver of chemokine mediated attraction for these immune suppressive cells infiltrating the tumor. Lastly, shorter overall survival was observed in these patients who have high CD70 expression compared to those with low CD70 expression on tumors. Taken together, the data suggest that CD70 plays a critical role in chemokine-mediated immune suppression and in GBM progression. Targeting GBM with a drug-conjugated CD70 antibody or CD70-CAR T-cells may improve the therapeutic efficacy for patients with GBM.