H3K27M突变型弥漫性中线胶质瘤中GD2-CAR T细胞治疗的纵向单细胞图谱揭示体液和细胞抗CAR免疫

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.