IMMU-03. Engineering novel synNotch-CAR T cells to precisely and effectively target medulloblastoma

Abstract While clinical trials involving chimeric antigen receptor (CAR) T cells have shown promise for treating pediatric brain tumors that have shown promise in clinical trials, factors such as antigen heterogeneity, on-target, off-tumor toxicity and an immunosuppressive microenvironment limit the efficacy of these treatments. To overcome these challenges, we adopted the synthetic notch receptor system and developed innovative synNotch-CAR T cell circuits. In this system a tissue-specific antigen primes the T cells to locally induce the expression of a CAR to completely kill the tumor cells. Here, we investigated the anti-tumor efficacy of synNotch-CAR T cells in patient-derived models of Group 3 medulloblastoma, the deadliest subtype of medulloblastoma. We utilized a synNotch receptor specific to brevican, a proteoglycan expressed solely in the central nervous system, for brain-specific priming. Human CD3+ cells were lentivirally transduced to express the synNotch receptor (α-BCAN) and an α-B7H3/IL13Ra2 CAR payload. α-BCAN synNotch-α-B7H3/IL13Ra2 CAR (B-SYNC) T cells were co-cultured with mCherry+ D425 and D283 tumor cells and real-time cell growth monitoring was performed using incucyte live imaging. Brevican-priming was accomplished either through brevican-­expressing K562 cells or tissue culture plates coated with recombinant human brevican. B-SYNC T cells completely killed the medulloblastoma cell line, D283, only when primed by K562 cells expressing BCAN. No killing was observed in the absence of priming cells. These cells also maintained killing of a more proliferative medulloblastoma cell line, D425, over a longer period than the constitutive -α-B7H3/IL13Ra2 CAR T cells. Furthermore, when cultured in brevican-coated plates, B-SYNC T cells killed D425 cells at a 17% faster rate than the constitutive -α-B7H3/IL13Ra2 CAR T cells. We are currently validating the superior efficacy of BSYNC cells in vivo using patient-­derived xenografts. Future studies are aimed at elucidating the mechanisms by which synNotch-CAR T cells outperform constitutive CAR T cells. Furthermore, we will investigate the anti-tumor efficacy of synNotch-CAR T cells in the more resistant, hypoxic medulloblastoma tumor microenvironment.