B7H3/IL13Rα2 bispecific armored CAR-T for recurrent/refractory glioblastoma: Safety, efficacy, and immune dynamics.

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Background: Recurrent/refractory glioblastoma (GBM) has an extremely poor prognosis with limited therapies. CAR-T therapy is innovative but hindered by antigen escape and insufficient durability; unclear early CAR-T trafficking/adaptation also limits its efficacy. We developed a fully human bispecific armored CAR-T targeting B7H3/IL13Rα2 with PK-guided multiple infusions and explored immune dynamics via single-cell multi-omics+TCR-seq. Methods: This 3+3 dose-escalation study (2.5, 5.0, 10, 20 million cells) enrolled 18-75-year-old recurrent/refractory GBM patients with ≥30% B7H3/IL13Rα2 expression (IHC). Patients received intracavity/intraventricular infusions via Ommaya catheter (PK monitoring guided dosing). Single-cell transcriptome+TCR-seq was performed on infused CAR-T and day-3 tumor cavity CSF to analyze immune dynamics. The primary objective was safety, and the secondary objectives were efficacy and the exploration of immune dynamics. Results: Two IDH-wildtype, MGMT-unmethylated patients completed 2.5 million-cell infusions, with no dose-limiting toxicities (DLTs) observed. Adverse events including fatigue, thrombocytopenia, abnormal liver function, and hypoalbuminemia were manageable without ICU admission, and the maximum grade of cytokine release syndrome (CRS) was 1, which resolved spontaneously within 3-5 days. Abundant central memory T cells and persistent high CAR-T copy numbers (≥3 weeks) were detected in the CSF, and per RANO2.0 criteria, the best overall responses were ≥30% tumor reduction in one patient (with 3-month progression-free survival) and stable disease (SD) in the second patient. Single-cell+TCR-seq showed infused CAR-T was predominantly CD8⁺/CD4⁺ T cells with heterogeneous phenotypes. Day-3 CSF had diverse immune populations; GSVA showed memory enrichment without dominant exhaustion. TCR-seq identified 188 infused-derived CAR-T cells (2.19% homing rate), mainly from cytotoxic/proliferating CD8⁺ and effector CD4⁺ T cells. Cytotoxic CD8⁺ T cells clonally expanded; proliferating CD8⁺ shifted to cytotoxic states; activated CD4⁺ transitioned to memory-like phenotypes. CSF CAR-T had stronger memory signatures (GSVA, p=2.48×10⁻⁹). Conclusions: B7H3/IL13Rα2 bispecific CAR-T with PK-guided low-dose infusions has favorable safety and preliminary efficacy in recurrent/refractory GBM, with robust CAR-T expansion/persistence. Single-cell analyses characterize immune dynamics, providing mechanistic insights for CAR-T optimization in solid tumors. Clinical trial information: NCT07193628 .