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.

• Tris-CAR-T cell injection (Biological): Autologous T cells transduced with a lentiviral vector to express a chimeric antigen receptor targeting CD44 and CD133. Each dose contains 1 × 10\^8 cells and is administered locally through an Ommaya reservoir into the tumor cavity or ventricle every 2 weeks, up to 12 injections over 6 months.
• Ommaya reservoir implantation (Procedure): An Ommaya reservoir is implanted into the tumor cavity or ventricle to enable local Tris-CAR-T cell administration and cerebrospinal fluid collection.
• Temozolomide (TMZ) (Drug): Standard temozolomide-based concurrent chemoradiotherapy followed by at least 6 cycles of adjuvant temozolomide according to institutional practice.
• radiotherapy (Radiation): Standard postoperative radiotherapy administered as part of concurrent chemoradiotherapy according to institutional practice.

Primary outcomes

• Progression-Free Survival (PFS) [From randomization through disease progression or death, assessed up to 2 years after the last administration.]: Time from randomization to first documented disease progression assessed according to RANO 2.0 or death from any cause, whichever occurs first.

Secondary outcomes

• Overall Survival (OS) [From randomization through death from any cause, assessed up to 2 years after the last administration.]: Time from randomization to death from any cause.
• 12-month Objective Response Rate (ORR) [At 12 months after randomization.]: Proportion of participants with complete response or partial response at 12 months as assessed by RANO 2.0.
• 12-month Disease Control Rate (DCR) [At 12 months after randomization.]: Proportion of participants with complete response, partial response, or stable disease at 12 months as assessed by RANO 2.0.
• Adverse Events (AEs) [From informed consent through the end of study follow-up, assessed up to 2 years after the last administration.]: Incidence, severity, and relationship of adverse events assessed according to NCI CTCAE v6.0 and coded using MedDRA.
• Serious Adverse Events (SAEs) [From informed consent through the end of study follow-up, assessed up to 2 years after the last administration.]: Incidence and description of serious adverse events, including death, hospitalization, life-threatening events, disability, or other medically important events.
• Cytokine Release Syndrome (CRS) [From first Tris-CAR-T cell infusion through the end of study follow-up, assessed up to 2 years after the last administration.]: Incidence and severity of cytokine release syndrome after Tris-CAR-T cell administration.
• Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) [From first Tris-CAR-T cell infusion through the end of study follow-up, assessed up to 2 years after the last administration.]: Incidence and severity of immune effector cell-associated neurotoxicity syndrome after Tris-CAR-T cell administration.

Other outcomes

• Cytokine Levels in Cerebrospinal Fluid or Tumor Cavity Fluid [From before Tris-CAR-T cell infusion through planned post-infusion follow-up time points, assessed up to 6 months after first infusion.]: Exploratory analysis of IL-2, IL-6, IFN-gamma, TNF-alpha, and other cytokine levels in cerebrospinal fluid or tumor cavity fluid and their association with efficacy and safety.

Sex: All, ages: 18 Years to 65 Years

Inclusion Criteria:

1. Voluntarily signs written informed consent.
2. 18 to 65 years of age, inclusive, male or female.
3. Newly diagnosed postoperative glioblastoma confirmed by histopathology or molecular pathology as WHO CNS grade 4 glioblastoma, IDH-wildtype, according to the 2021 WHO Classification of Central Nervous System Tumors.
4. Tumor tissue positive for CD44 or CD133 antigen expression by immunohistochemistry, defined as CD44 or CD133 staining positive in at least 1% of tumor cells, regardless of staining intensity.
5. At least one preoperative measurable lesion meeting RANO 2.0 criteria outside the radiation field, with the longest diameter of the measurable lesion no greater than 3 cm by imaging assessment.
6. No prior radiotherapy and no prior treatment with temozolomide, bevacizumab, or other anti-tumor therapy for glioblastoma.
7. Confirmed by the investigator to be suitable for Ommaya reservoir implantation.
8. Adequate venous access for peripheral blood mononuclear cell collection.
9. Karnofsky Performance Status (KPS) score of at least 70 within 1 week before PBMC collection and within 4 days before Tris-CAR-T cell infusion.
10. Expected survival of at least 3 months.
11. Adequate bone marrow, hepatic, renal, coagulation, and oxygenation function within 1 week before PBMC collection and within 1 week before Tris-CAR-T cell infusion, including HGB at least 100 g/L, WBC at least 3.5 × 10\^9/L, ANC at least 1.5 × 10\^9/L, PLT at least 100 × 10\^9/L, lymphocyte count greater than 1 × 10\^9/L, ALT and AST no more than 2.5 × ULN, TBIL no more than 1.5 × ULN, creatinine no more than 1.5 × ULN, PT or INR no more than 1.5 × ULN, APTT no more than 1.5 × ULN, and resting oxygen saturation without oxygen at least 95%.
12. Left ventricular ejection fraction greater than 50% and electrocardiogram QTcF no more than 480 msec within 14 days before PBMC collection and within 14 days before Tris-CAR-T cell infusion.
13. For female participants of childbearing potential, negative pregnancy tests within 2 weeks before PBMC collection and within 1 week before the first CAR-T cell infusion. Participants of childbearing potential, male or female, must agree to use effective medical contraception from screening through 2 years after the last cell infusion.
14. Willing and able to comply with protocol-specified visits, study treatment, laboratory tests, and other study-related requirements.

Exclusion Criteria:

1. Autologous CAR-T cell manufacturing cannot be completed or the CAR-T cell product does not meet clinical use requirements.
2. Highly allergic constitution, history of severe allergy, or allergy to related cell products.
3. Receipt of approved or investigational anti-tumor therapy within 2 weeks before PBMC collection or within 5 half-lives of the therapy, whichever is longer, including but not limited to chemotherapy, radiotherapy, surgery other than diagnostic surgery, targeted therapy, cell infusion therapy, hormone therapy other than hormone replacement therapy, or traditional Chinese medicine with a clear anti-tumor indication; a 1-week washout is acceptable for traditional Chinese medicine.
4. Severe clinical infection greater than Grade 2 by NCI CTCAE v6.0 within 2 weeks, uncontrolled active infection, or unexplained fever before PBMC collection or Tris-CAR-T cell infusion.
5. Prior anti-tumor treatment-related adverse reactions have not recovered to Grade 1 or lower by NCI CTCAE v6.0, except alopecia, skin pigmentation, leukoplakia, or other findings judged by the investigator to pose no safety risk.
6. Tumor lesion metastasis to the brainstem or spinal cord.
7. Serious neurological diseases other than the brain tumor, such as meningitis, encephalitis, myelitis or demyelinating disease, myasthenic syndrome or myasthenia gravis, amyotrophic lateral sclerosis, spinal muscular atrophy, nerve palsy, or uncontrolled epilepsy.
8. Myocardial infarction, coronary artery bypass grafting, coronary angioplasty, coronary stent implantation, pulmonary embolism, new arterial or deep venous thrombosis, stroke, or transient ischemic attack within 6 months, excluding brain tumor bleeding or brain tumor surgery-related bleeding.
9. Heart failure NYHA class 2 or higher, myocarditis, cardiomyopathy, pericarditis, cardiac valve morphological abnormality Grade 2 or higher, unstable coronary heart disease, poorly controlled hypertension with systolic blood pressure at least 150 mmHg or diastolic blood pressure at least 100 mmHg, or severe arrhythmia or conduction abnormality requiring medication.
10. Severe respiratory disease requiring treatment, such as pneumonia, chronic bronchitis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, interstitial lung disease, or respiratory failure.
11. Hepatitis requiring treatment, uncontrolled or unstable liver function impairment, or diabetes mellitus not controlled with medication.
12. Severe urinary system disease or abnormality requiring treatment, such as proteinuria, glomerulonephritis, or renal function abnormality.
13. Primary immunodeficiency disease, autoimmune disease requiring medication, such as Crohn disease, ulcerative colitis, rheumatoid arthritis, or systemic lupus erythematosus, or history of neurological autoimmune disease such as multiple sclerosis or Parkinson disease.
14. Receiving or requiring long-term immunosuppressive therapy, except physiologic systemic corticosteroid replacement equivalent to prednisone 10 mg/day or less, short-term corticosteroids for no more than 7 days for prevention or treatment of non-autoimmune allergic disease, or topical, intra-articular, intranasal, inhaled, or other local glucocorticoids.
15. Prior allogeneic bone marrow transplantation or organ transplantation, or awaiting organ transplantation.
16. HBsAg positive with HBV DNA positive, HCV antibody positive with HCV RNA positive, Treponema pallidum antibody positive, or HIV antibody positive.
17. Prior gene therapy or cell therapy.
18. Pregnant or lactating female.
19. History of malignancy other than glioma within 5 years, except adequately treated carcinoma in situ, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, localized prostate cancer, or stage I cervical cancer judged by the investigator to have very low risk of recurrence.
20. Any other disease, abnormal test result, psychiatric factor, or social factor that may affect compliance with protocol procedures, place the participant at high risk when receiving the investigational product, or interfere with interpretation of study results, making the participant unsuitable for this clinical study in the investigator's judgment.

• Results publication: Zhai Y, Li G, Pan C, Yu M, Hu H, Wang D, Shi Z, Jiang T, Zhang W. The development and potent antitumor efficacy of CD44/CD133 dual-targeting IL7Ralpha-armored CAR-T cells against glioblastoma. Cancer Lett. 2025 Apr 1;614:217541. doi: 10.1016/j.canlet.2025.217541. Epub 2025 Feb 12. PMID 39952598

• Wei Zhang, MD, PhD, Principal investigator, Beijing Tiantan Hospital
• Tao Jiang, MD, PhD, Study chair, Beijing Neurosurgical Institute