Abstract GD2-CAR T cell therapy has demonstrated clinical benefit in a high fraction of patients with H3K27M-mutant diffuse midline glioma (DMG), but the durability of response is limited in some patients. To define mechanisms underlying loss of response and immune adaptation during repeated CAR T infusions, we performed longitudinal single-cell RNA and TCR sequencing of over 1.4 million cells from apheresis, CAR T products, and serial cerebrospinal fluid (CSF) samples collected from 13 DMG patients enrolled in the Arm A of Phase I trial of GD2-CAR T cell therapy (NCT04196413). Our analysis showed that GD2-CAR T cells exhibited poor persistence in vivo, with minimal detection after six infusion cycles. Endogenous lymphocytes, rather than CAR T cells, dominated the CSF immune compartment over time. Regulatory T cells were significantly enriched in non-responders, suggesting a suppressive immune milieu that limits CAR T activity. Among four patients who initially achieved sustained responses for over 12 months, three eventually relapsed after repeated infusions. Flow cytometry detected presence of anti-CAR antibodies in plasma and CSF these three patients, accompanied by an increase in CSF B cells at the time of relapse, indicating a humoral anti-CAR immune response. Complementing this, single-cell TCR-seq revealed that endogenous T cell repertoires stabilized toward the end of treatment in these relapsed responders, with hyper-expanded clonotypes exhibiting activated, cytotoxic, and exhausted transcriptional programs. In contrast, two durable responders maintained a dominant tissue-resident memory-like T cell program, which is associated with sustained disease control. Altogether, we generated a comprehensive longitudinal collection of single-cell sequencing and other omics data, and identified co-existing humoral and cellular mechanisms of anti-CAR immunity that emerge during prolonged GD2-CAR T cell therapy. These findings illuminate correlates of response and resistance and underscore the immunogenic potential of CAR constructs. Future approaches to reduce host immunoreactivity could improve CAR T cell efficacy and durability in solid tumors. Citation Format: Yiyun Chen, Kun-Wei Song, Moksha H. Desai, Ying-Wen Huang, Nadya Iswari, Zachary J. Ehlinger, Hossein Daghagh, Maximilian R. Koch, Kevin Reynolds, Kelvin C. Mo, Kristin C. Tsui, Skyler Rietberg, Mark P. Hamilton, Snehit Prabhu, Sonia Partap, Jasia Mahdi, Emily Egeler, Steven A. Feldman, Sabine Heitzeneder, Sean A. Yamada-Hunter, Elena Sotillo, Bita Sahaf, Zinaida Good, Michelle Monje, Sneha Ramakrishna, Crystal L. Mackall. Longitudinal single-cell atlas of GD2-CAR T cell therapy in H3K27M-mutant diffuse midline glioma identifies humoral and cellular anti-CAR immunity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6463.
Chen et al. (Fri,) studied this question.