Abstract 7123: Unravelling temporal dynamics of GD2-CAR T cell therapy in H3K27M-mutant diffuse midline glioma through comprehensive single-cell and multi-omic profiling

Abstract H3K27M-mutant diffuse midline gliomas (DMG) are aggressive pediatric brain tumors with dismal prognoses and no effective treatments. GD2, a disialoganglioside overexpressed on DMG cells, is a promising target for chimeric antigen receptor (CAR) T cell therapy. In Arm A of the Phase I clinical trial (NCT04196413), 12 patients (11 on study) received GD2-CAR T cells through an initial intravenous (IV) infusion, and if patients had clinical or radiographic improvement, they subsequently received intracerebroventricular (ICV) administrations. This regimen achieved clinical benefit in ten patients, with >50% tumor volumetric reductions in 5 patients and a durable ongoing complete response in one patient (Majzner, Ramakrishna et al. Nature 2022; Monje et al. Nature 2024). However, disease progression ultimately occurred in 11 patients underscoring challenges in therapeutic optimization. To elucidate mechanisms underlying treatment responses and resistance, we performed a comprehensive multi-omic analysis, integrating single-cell RNA and TCR sequencing, mass cytometry, and clinical and molecular datasets. Sequencing 1, 062, 398 cells from patient apheresis, CAR T products, and cerebrospinal fluid (CSF) samples collected longitudinally at pre-defined trigger events, we reconstructed the dynamic landscape of immune and tumor cell populations in 12 DMG patients. Our analysis revealed profound CAR T cell infiltration into the CSF after treatment, accompanied by increases in endogenous T cell, mast cell, and monocyte populations and a relative decrease in microglia evident in CSF. This works sought to deeply characterize the lymphocyte profiles in these samples. Inflammatory and proliferation pathways were elevated in CAR T cells at early post-ICV time points, transitioning into an NK-like state and TGF-β receptor signaling at later stages. TCR clonal tracking identified persistent CAR T clones, alongside endogenous T cell activation and expansion. Moreover, non-responders to the therapy showed elevated Treg signatures in the CSF. Using CellChat-based cell-cell communication analysis, we uncovered differential cytokine-receptor interactions in responders versus non-responders, offering novel insights into immune-tumor interactions in the context of CAR T cell therapy. By contextualizing single-cell data within multi-omic frameworks and clinical outcomes, this study advances our understanding of the GD2-CAR T cell therapy, the tumor microenvironment, and biomarkers of response and resistance. These findings provide critical insights for optimizing CAR T cell therapies in CNS malignancies and inform the development of next-generation immunotherapies. Citation Format: Yiyun Chen, Moksha H. Desai, Zachary J. Ehlinger, Kun Wei Song, Skyler Rietberg, Hossein Daghagh, Mark P. Hamilton, Snehit Prabhu, Sonia Partap, Jasia Mahdi, Elena Sotillo, Bita Sahaf, Sneha Ramakrishna, Zinaida Good, Michelle Monje, Crystal Mackall. Unravelling temporal dynamics of GD2-CAR T cell therapy in H3K27M-mutant diffuse midline glioma through comprehensive single-cell and multi-omic profiling [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 7123.