Abstract 6114: Engineering novel synNotch CAR T cells to precisely and effectively target medulloblastoma
Authors: Maggie Colton Cove, Senthilnath Lakshmanachetty, Milos Simic, Robert Zhu, Chanelle Shepherd, Lia Cardarelli, Sachdev Sidhu, Olga Troyanskaya, Wendell Lim, Hideho Okada
Journal: Cancer Research (2025)

Abstract Background: While clinical trials involving chimeric antigen receptor (CAR) T cells have shown promise for treating pediatric brain tumors, 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 synthetic notch receptor system and developed innovative synNotch-CAR T cell circuits. In this system, the first antigen which is expressed exclusively on the brain, 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. Methods: 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 stimulated with CD3/CD28 beads for 24 hours and lentivirally transduced to express the synNotch receptor (α-BCAN) and a 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 for 4 days 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. Tumor cell growth was measured by mCherry fluorescent signal. Results: 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 an extended 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. Conclusions: B-SYNC T cells show superior cell-killing to constitutive -α-B7H3/IL13Ra2 CAR T cells in in vitro models of Group 3 medulloblastoma. We are currently validating this finding 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. Citation Format: Maggie Colton Cove, Senthilnath Lakshmanachetty, Milos Simic, Robert Zhu, Chanelle Shepherd, Lia Cardarelli, Sachdev Sidhu, Olga Troyanskaya, Wendell Lim, Hideho Okada. Engineering novel synNotch CAR T cells to precisely and effectively target medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6114.