Intracerebroventricular bivalent CAR T cells targeting EGFR and IL-13Rα2 in recurrent glioblastoma: a phase 1 trial

Abstract

Glioblastoma (GBM) is the most common primary brain cancer in adults and carries a median overall survival (OS) of 12-15 months. Effective therapy for recurrent GBM (rGBM) following frontline chemoradiation is a major unmet medical need. Here we report the dose escalation and exploration phases of a phase 1 trial investigating intracerebroventricular delivery of bivalent chimeric antigen receptor (CAR) T cells targeting epidermal growth factor receptor (EGFR) epitope 806 and interleukin-13 receptor alpha 2 (IL-13Rα2), or CART-EGFR-IL13Rα2 cells, in patients with EGFR-amplified rGBM. Primary endpoints included dose-limiting toxicity, determination of the maximum tolerated dose and recommended dose for expansion, and occurrence of adverse events. Secondary endpoints included objective radiographic response, duration of response, progression-free survival and OS. A total of 18 patients received CART-EGFR-IL13Rα2 cells. The maximum tolerated dose was determined to be 2.5 × 10⁷ cells. Of the 18 patients, 10 (56%) experienced grade 3 neurotoxicity; none had grade 4-5 neurotoxicity. Of 13 patients, 8 (62%) with measurable disease at the time of CAR T cell infusion experienced tumor regression, with one confirmed partial response by Modified Response Assessment in Neuro-Oncology criteria (objective radiographic response, 8%; 90% confidence interval, 0-32%) and one patient with ongoing durable stable disease lasting over 16 months. Median progression-free survival was 1.9 months (90% confidence interval, 1.1-3.4 months), and median OS was not yet reached at the time of data cut-off (median follow-up time, 8.1 months). These findings indicate that intracerebroventricular delivery of bivalent CART-EGFR-IL13Rα2 is feasible and appears safe. CART-EGFR-IL13Rα2 cells are bioactive and exhibit a signal of antitumor effect in rGBM. ClinicalTrials.gov registration: NCT05168423 .

Extended Data Fig. 1. Immunofluorescence images of representative pre-infusion tumor samples.

Patient slides demonstrated a range of target staining, including tissue with subjective positivity for both targets (16321–04, 16321–40, 16321–23, 16321–52), for EGFR (16321–18), and for IL13Ra2 (16321–11). Given the trial inclusion criterion of EGFR amplification, the negative staining for EGFR seen in 16321–11 is a relevant example of CAR target spatial heterogeneity. All images taken at 20x magnification. Experiments were performed in duplicate.

Extended Data Fig. 2. Individual CSF pharmacokinetics for dose level −1

CAR copies per ug of genomic DNA (Top). CAR copies per mL of CSF (Bottom).

Extended Data Fig. 3. Individual CSF pharmacokinetics for dose level 1

CAR copies per ug of genomic DNA (Top). CAR copies per mL of CSF (Bottom).

Extended Data Fig. 4. Individual CSF pharmacokinetics for dose level 2

CAR copies per ug of genomic DNA (Top). CAR copies per mL of CSF (Bottom).

Extended Data Fig. 5. Fold change in CAR copies/ug DNA in the CSF from Day +1 to Day +7 by dose level

Fold change in CAR copies/ug DNA in the CSF from Day +1 to Day +7 by dose level (two-sided p=0.107, Wilcoxon test).

Extended Data Fig. 6. Mean fold increase of key inflammatory cytokines in the CSF from Day 0 through Day 28 across all patients by dose level.

Mean fold increase of cytokines interferon-gamma (IFNg), interleukin-2 (IL2), interleukin-6 (IL6), and tumor necrosis factor alpha (TNFa) in the CSF from Day 0 through Day 28 across all patients by dose level (n=6 per dose level). Data are presented as mean values +/− SEM.

Extended Data Fig. 7. T1 post-contrast (top panels) and T2/FLAIR (bottom panels) MRI images for the 8 patients with measurable disease who experienced any degree of tumor regression following CAR T cell infusion.

Timepoints shown include (1) the post-operative MRI scan taken on post-operative day 1 following the surgery that was performed for maximal safe tumor resection and Ommaya reservoir placement, (2) the immediate pre-CART MRI scan taken within 1–2 days prior to CAR T cell infusion, and (3) the 1-month post-CART MRI scan. No anticancer therapies were administered between scan 1 and 2, and no anticancer therapies other than CART-EGFR-IL13Ra2 cells were administered between scan 2 and scan 3. In Patient-07, an increase in enhancing tumor on the 1-month MRI was followed by spontaneous regression on the 2-month MRI, consistent with pseudo-progression.

Extended Data Fig. 8. Individual CSF pharmacokinetics patients who underwent retreatment with CAR T cells

CAR copies per ug of genomic DNA (Top). CAR copies per mL of CSF (Bottom).

Figure 1.. Study overview.

a, Schema of patient treatment. b, CONSORT diagram of patient enrollment.

Figure 2.. Toxicity overview.

Incidence (number of patients) and grade of treatment-emergent AEs occurring in >20% of patients (n=18), including neurotoxicity and CRS.

Figure 3.. CAR T cell pharmacokinetics and cytokines in the CSF of all patients (n=18).

a, Summary of longitudinal CAR T cell pharmacokinetics in CSF by dose level (data are presented as mean values +/− SEM). b, Summary of longitudinal CAR T cell pharmacokinetics in peripheral blood by dose level (data are presented as mean values +/− SEM). c, Comparison of the AUC_{0–28 days} in CSF by dose level. The box represents the interquartile range (IQR), showing the middle 50% of the data, and the center line in each box represents the median value. The upper whisker extends the 75^th percentile value plus 1.5 times the IQR, and the lower whisker extends to the 25^th percentile minus 1.5 times the IQR. d, Mean fold increase of interferon-gamma (IFNg), interleukin-2 (IL2), interleukin-6 (IL6), and tumor necrosis factor alpha (TNFa) in the CSF from Day 0 through Day 28 across all patients. Data are presented as mean values +/− SEM.

Figure 4.. Efficacy measures.

a, Waterfall plot (n=13 patients with measurable disease at time of CAR T cell infusion) depicting best tumor regression compared to baseline and present at the day +28 MRI scan and/or later timepoints (DL-1, dose level −1; DL1, dose level 1; DL2, dose level 2). b, Swimmer plot (n=18) depicting patient survival. c, T1 post-contrast MRI images for Patient-28 (top panel), who experienced a confirmed partial response by mRANO criteria, and Patient-26 (bottom panel), who experienced marked tumor regression of a large enhancing tumor in the splenium of the corpus callosum but had tumor progression one month later. d, Progression-free survival (n=18). e, Progression-free survival in patients treated in dose level −1 (5.0 × 10⁶ cells, n=6) vs. patients treated in dose levels 1 and 2 (1.0 × 10⁷ cells, 2.5 × 10⁷ cells, n=12). Two-sided log-rank p value = 0.15 (no adjustment for multiple comparisons). f, Pre- and post-CAR T cell histology for Patient-52, who experienced radiographic progression and underwent repeat craniotomy for tumor resection on day +117 following infusion of 5.0 × 10⁶ CAR T cells. Before CAR T cell infusion, the histology demonstrated recurrent glioma and reactive changes as well as areas of necrosis consistent with radiation-related changes. Activated microglia/macrophages are highlighted by a CD163 stain. In contrast, in the post-infusion specimen, macrophages have epithelioid foamy cytology and are packed with GFAP+ material. The macrophages are present in sheets where the tumor has been obliterated and in pools within the recurrent tumor. The areas with macrophages are rich in CD8+ T cells, which are also scattered in the tumor. GFAP is positive in the glial areas, but also positive in the macrophages, indicative of the macrophage clearance activity. The foamy macrophages and their GFAP+ content are shown at higher power in the lowest three images. An area of gliotic parenchyma with infiltrating tumor in the lower right allows differentiation of the CD68 and GFAP stains. A 50-micron-wide inset shows a single cell in the GFAP image. Staining was performed once on these specimens and not repeated.