Dual-Targeting CAR-NK Cells for Recurrent/Progressive Glioblastoma and High-Grade Glioma

Rationale: GBM/HGG is molecularly heterogeneous and may evade single-antigen immunotherapy via antigen loss or heterogeneous antigen expression. This study evaluates a dual-target CAR-NK strategy that can recognize two tumor-associated antigens selected from IL13Rα2, EGFR/EGFRvIII, and B7-H3 (CD276).

Target assessment and cohort assignment: At screening, archived and/or fresh tumor tissue (from clinically indicated resection or biopsy) will be evaluated by immunohistochemistry (IHC) for IL13Rα2, EGFR, and B7-H3, and by molecular testing (e.g., RT-PCR or NGS) for EGFRvIII when applicable. Participants will be assigned to one of three biomarker-defined cohorts based on the two highest-priority antigens detected above protocol-defined thresholds. An internal review committee may recommend prioritizing one construct for later expansion based on emerging safety/feasibility/activity signals.

Investigational product: The investigational products are off-the-shelf allogeneic CAR-NK cells manufactured from a standardized NK-cell source (e.g., iPSC-derived NK cells or a qualified NK master cell bank) and genetically modified to express a tandem (dual-target) CAR. The constructs include a built-in safety switch and may include an NK-support cytokine module (e.g., IL-15) to enhance persistence. The exact dual-target construct used for each participant depends on the screening antigen profile.

Route of administration: CAR-NK cells will be delivered locoregionally to the surgical cavity wall and/or into the ventricular system via a neurosurgically placed catheter/reservoir, to maximize exposure at the tumor site while limiting systemic exposure.

Dose escalation and expansion: Each biomarker-defined cohort follows a modified 3+3 dose-escalation schema with up to three dose levels (e.g., 1×10^7, 3×10^7, and 1×10^8 CAR-NK cells per infusion). Dose-limiting toxicities (DLTs) will be assessed during the first 28 days after the first infusion. After an RP2D is identified within a cohort, an expansion stage will further characterize safety and preliminary activity and may allow repeat infusions at the RP2D.

Follow-up: Participants will be monitored closely for adverse events including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).

Tumor response will be assessed with MRI using RANO criteria at regular intervals.

Long-term follow-up for gene-modified cell therapy safety will be conducted per applicable guidance.

Inclusion criteria

• Age 18 to 75 years at the time of consent.
• Histologically confirmed glioblastoma (WHO grade 4) or diffuse high-grade glioma (WHO grade 3 or 4) that is recurrent or progressive after standard therapy.
• Planned clinically indicated tumor resection or stereotactic biopsy (or availability of adequate archived tumor tissue) to support antigen testing and locoregional catheter placement.
• Tumor demonstrates expression of at least two of the following antigens above protocol-defined thresholds: IL13Rα2, EGFR (wild-type) and/or EGFRvIII, B7-H3 (CD276).
• Karnofsky Performance Status (KPS) ≥ 60.
• Adequate organ function (hematologic, renal, hepatic) as defined by protocol laboratory criteria.
• Ability to undergo brain MRI with contrast (unless contraindicated and alternative imaging is permitted).
• Negative pregnancy test for women of childbearing potential; agreement to use effective contraception during study participation and for a protocol-defined period after infusion.
• Ability to understand and willingness to sign informed consent.

Exclusion criteria

• Active, uncontrolled infection (including uncontrolled bacterial, viral, or fungal infection).
• Known HIV infection with uncontrolled viral load; active hepatitis B or hepatitis C with detectable viral load (unless permitted per protocol).
• Clinically significant autoimmune disease requiring systemic immunosuppression within the past 6 months.
• Requirement for high-dose systemic corticosteroids (e.g., >4 mg/day dexamethasone equivalent) within 7 days prior to lymphodepletion/infusion (physiologic replacement permitted).
• Prior gene-modified cellular therapy (e.g., prior CAR-T/CAR-NK) within 6 months, or prior therapy targeting IL13Rα2, EGFR/EGFRvIII, or B7-H3 where residual engineered cells could confound safety assessments.
• Diffuse leptomeningeal disease as the only site of disease, or anatomy that precludes safe catheter placement (unless specifically allowed by protocol).
• Uncontrolled seizures despite optimal medical therapy.
• Clinically significant cardiovascular disease (e.g., recent myocardial infarction, uncontrolled arrhythmia) that would increase risk with lymphodepletion or infusion procedures.
• Pregnant or breastfeeding.
• Any condition that, in the investigator's judgment, would make the participant unsuitable for the study or could interfere with protocol adherence.