Tris-CAR-T Cell Therapy Targeting CD44 and CD133 in Newly Diagnosed Glioblastoma

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor in adults, characterized by marked intratumoral heterogeneity, an immunosuppressive tumor microenvironment, and a high risk of recurrence. Despite maximal safe surgical resection followed by standard temozolomide-based chemoradiotherapy, the prognosis of patients with newly diagnosed GBM remains poor. Novel therapeutic approaches are needed to improve disease control and long-term survival.

Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a promising immunotherapeutic strategy for GBM. However, its efficacy may be limited by heterogeneous antigen expression, antigen escape, and inadequate T-cell persistence within the tumor microenvironment. To address these challenges, our research group has developed autologous CD44/CD133 dual-target Tris-CAR-T cells. These T cells are genetically modified using a lentiviral vector to express a dual-targeting CAR recognizing CD44 and CD133, two antigens associated with GBM and glioma stem-like cell populations. The CAR construct incorporates a truncated interleukin-7 receptor alpha (IL-7Rα) intracellular domain between the CD28 costimulatory domain and the CD3ζ signaling domain, designed to enhance T-cell survival, persistence, and sustained antitumor activity [1].

Preclinical studies conducted by our group demonstrated that CD44/CD133 dual-targeting IL-7Rα-armored CAR-T cells exhibited potent and sustained antitumor activity across multiple experimental GBM models, including patient-derived glioma stem cell cultures, intracranial xenograft models, patient-derived xenograft models, and patient-derived glioblastoma organoids. Locoregional administration of these CAR-T cells resulted in sustained tumor control in preclinical intracranial GBM models, providing a rationale for further clinical investigation.

The clinical development of this therapeutic approach has been supported by previous and ongoing studies conducted at Beijing Tiantan Hospital, Capital Medical University. An investigator-initiated Phase I clinical trial (NCT05577091) has investigated the safety and preliminary efficacy of locoregionally administered autologous Tris-CAR-T cells in patients with recurrent GBM. In addition, an ongoing Phase I/II clinical trial (NCT07318818) is evaluating P134 cells, a CD44/CD133 dual-targeting CAR-T cell product, in patients with recurrent GBM. These clinical investigations provide a foundation for extending the evaluation of this therapeutic strategy to patients with newly diagnosed GBM.

The present study investigates the feasibility, safety, and preliminary efficacy of incorporating locoregional Tris-CAR-T cell therapy into the postoperative treatment strategy for newly diagnosed, IDH-wildtype GBM. In the experimental arm, an Ommaya reservoir will be implanted into the tumor resection cavity or ventricular system approximately 4 to 6 weeks after surgery. Tris-CAR-T cells will be administered locally through the Ommaya reservoir at a dose of 1 × 10^8 CAR-positive T cells per administration, with treatment repeated every 2 weeks. A maximum of 12 administrations will be permitted within 6 months, according to the study protocol and individual treatment tolerance.

Following Tris-CAR-T cell treatment, participants in the experimental arm will receive standard concurrent radiotherapy and temozolomide chemotherapy, followed by adjuvant temozolomide for at least 6 cycles, as specified in the study protocol. If tumor progression is confirmed during Tris-CAR-T cell therapy, further Tris-CAR-T administration will be discontinued, and participants will transition to standard temozolomide-based chemoradiotherapy. Participants in the active comparator arm will receive standard postoperative concurrent chemoradiotherapy followed by adjuvant temozolomide.

Tumor response and disease progression will be assessed using magnetic resonance imaging (MRI) according to Response Assessment in Neuro-Oncology (RANO) 2.0 criteria. Safety evaluations will include treatment-related adverse events, serious adverse events, cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), laboratory parameters, vital signs, neurological examinations, and Karnofsky Performance Status (KPS). The study will assess whether the addition of locoregional Tris-CAR-T cell therapy can improve clinical outcomes while maintaining an acceptable safety profile.

Exploratory investigations will evaluate treatment-associated immune responses and potential biomarkers of efficacy and toxicity. Where feasible, cerebrospinal fluid and/or tumor cavity fluid will be collected to characterize cytokine profiles and their associations with clinical outcomes. These investigations may provide insights into the local immune responses induced by intracranial CAR-T cell administration and support future biomarker-guided therapeutic strategies.

Long-term follow-up will be conducted in accordance with the study protocol and applicable regulatory requirements for genetically modified cellular therapies. This study is expected to provide prospective clinical evidence regarding the feasibility and potential clinical benefit of CD44/CD133 dual-target Tris-CAR-T cell therapy in newly diagnosed GBM and to inform further development of CAR-T-based immunotherapeutic strategies for malignant gliomas.