IMMU-21. SEQUENTIAL TWO-RECEPTOR PRIMING CAR SYSTEM TO OVERCOME HETEROGENEOUS ANTIGEN EXPRESSION

Abstract

Abstract Heterogeneous expression of target antigens allows tumor escape from chimeric antigen receptor-transduced T-cell (CART) therapy targeting a single antigen. While epidermal growth factor receptor (EGFR)vIII represents a glioblastoma (GBM)-specific antigen, its expression is heterogeneous within the tumor. On the other hand, most other antigens expressed more uniformly in GBMs are non-mutated, glioma-associated antigens (GAAs), such as EphA2. Although these GAAs are not expressed in the normal brain, they are expressed at low levels in other normal organs. As a way to safely target GAAs in the tumor without attacking normal cells expressing the same GAAs outside of the brain, we adapted a novel synthetic Notch (synNotch) receptor system, and established a “prime and kill” sequential two-receptor CAR circuit: the first is a transcriptional CAR against EGFRvIII, and the second is a CAR against a GAA (e.g. EphA2). When the first CAR binds to EGFRvIII, it induces the expression of the second CAR against GAA. While the first CAR does not trigger the cytotoxic function itself, the second CAR mediates the cytotoxicity upon recognition of the target GAA. We validated this system in vitro using the mixture of U87 GBM cells and those transduced with EGFRvIII. SynNotch CART effectively lysed both EGFRvIII+ and EGFRvIII-negative U87 cells when they are mixed, but never lysed EGFRvIII-negative cells in the absence of EGFRvIII+ cells. SynNotch CART cells exhibited precise local effect in vivo, sparing EGFRvIII-negative subcutaneous tumor while efficiently clearing intracranial EGFRvIII heterogeneous tumor. In the orthotopic GBM6 model, which is a patient-derived xenograft with heterogeneous expression of EGFRvIII, intravenous infusion of synNotch CART resulted in long-term (over 50 days) survival and eradication of the heterogeneous tumor in 6 of 6 mice, while the conventional anti-EGFRvIII CART failed to do so. These data provide a strong basis for developing synNotch CART therapy for GBM.