Article Figures & Data
Figures
- Figure 1.
Treatment schema and IL13-zetakine+ CTL manufacturing. A, four weekly cycles of intracavitary cell doses were administered after enrolled patients experienced recurrence and underwent tumor excision with placement of a Rickham catheter. Patients had one week of rest for brain imaging between cycles 2 and 3. B, schematic of the manufacturing process, with day of each step(s) and in-process analyses indicated. CRA, chromium release assay; GCV, ganciclovir; Myco, mycoplasma; OKT3, anti-CD3 antibody used to activate T cells. C, characterization of the three cell products administered to patients with recurrent GBM. Depicted from left to right: Southern blots of T-cell genomic DNA using a hygromycin-specific probe (detecting HyTK selection/suicide fusion gene) showing existence of single bands as indicated by arrows; flow cytometric analysis of surface CAR expression using anti-IL13 antibody, and of T-cell markers using antibodies specific for TCR-αβ, CD3, CD8, CD28, CD45RO, CD45RA, CD62L, CD69, CD95, NKG2D, and TIM-3 where isotype control staining is indicated with the open histograms or quadrant placement; ability of CTL clones to lyse IL13Rα2+ targets U251T and Daudi-13Rα2, but not nonengineered Daudi cells, determined in a 4-hour 51Cr-release assay; and ganciclovir sensitivity using a flow cytometry–based assay for viable cell numbers after 14 days of culture with either rHuIL-2 or rHuIL-2 + ganciclovir.
- Figure 2.
Tumor IL13Rα2 expression for each patient. A, immunohistochemical staining of IL13Rα2 on paraffin-embedded primary patient-derived brain tissues. Sections were scored blindly by a neuropathologist for staining intensity (0, not detected; 1+, low; 2+, moderate; 3+, high), and percentages of positive cells are indicated. B, quantification of DAB intensity (lumen × pixel2) for dense tumor regions (>60% tumor, red outline). C, gene expression of IL13Rα2 mRNA levels evaluated by qPCR using Taqman gene expression assay.
- Figure 3.
Brain MRIs detected inflammation following IL13-zetakine+ CTL administration. MRI for (A) UPN028, (B) UPN033, and (C) UPN031 before and after intracranial administration of IL13-zetakine+ CD8+ CTL. MRI images are shown before-therapy, i.e., post surgical resection and prior to T-cell administration (within 12 days); and post-therapy, i.e., following cycles 1 and 2 (within 10 days), following cycles 3 and 4 (within 5 days), and 60 days or more after the last T-cell treatment. Top rows, post Gd T1-weighted images demonstrating changes in contrast uptake at the site of T-cell injections over treatment duration. Bottom rows, FLAIR images without Gd highlighting progression of cerebral edema following T-cell therapy. White arrow, site of tumor recurrence for UPN033.
- Figure 4.
Effect of CAR T-cell therapy on IL13Rα2 expression by the tumor of UPN031. Primary patient-derived brain tumor tissue from UPN031 was excised at day 8 (PRE Therapy) and day 184 (POST Therapy) according to the timeline depicted in Supplementary Fig. S2B. A, flow cytometric analysis of IL13Rα2 surface expression by freshly dissociated tumor cells using anti-IL13Rα2 antibody. Percentage of immunoreactive cells (gray) above isotype control (black line) is indicated in each histogram. B, gene expression of IL13Rα2 mRNA levels evaluated by qPCR using Taqman gene expression assay. C, IHC staining (IL13Rα2-specific DAB with hematoxylin counterstain (top) and histograms of IL13Rα2-specific DAB staining density (bottom). Left, proportion of pixels with IL13Rα2-specific DAB staining between 0 (clear) and 255 (opaque). Right, cumulative proportions of pixels with IL13Rα2-specific DAB staining, with median values (SD50) indicated.
- Figure 5.
MRI of UPN033 shows increased necrotic tumor volume following administration of IL13-zetakine+ CD8+ T-cell clones. A, T1-weighted post-gadolinium MRI before and after the 5 daily infusions of autologous IL13 (E13Y)-zetakine+/HyTK+ CD8+ CTL. B, MRS analysis of enhancing volume (indicative of neuroinflammation) and necrotic volume at the recurrent site following retreatment with T cells. Lack of tumor recurrence at the original treatment site (right occipital) and increase in necrosis at the recurrent site (left corpus callosum, white circles in A) are highly suggestive of therapeutic activity. C, single voxel MR spectroscopy with a pane over the lesion medial to the atrium of the left lateral ventricle on day 314. Choline (Cho), creatine (Cr), N-acetyl-l-aspartate (NAA), and lactate/lipid (Lac/Lip) peaks are indicated.
Tables
- Table 1.
Safety and tolerability
Patient T-cell doses Maximum tolerated T-cell dose Cumulative T-cell dose Adverse eventa Survival post relapse (months) Intracavitary delivery UPN028 11 108 9.6 × 108 Headaches 10.3 UPN031 12 108 10.6 × 108 Neurologic – Otherb 8.6 UPN033 12 108 10.6 × 108 None 13.9 Intratumoral delivery UPN033c 5 108 3.75 × 108 Low WBC 13.9d Headache Fatigue ↵aOnly events of Grade 3 or higher, according to the NCI Common Toxicity Criteria, with possible or higher attribution to the T cell administration are reported.
↵bShuffling gait and tongue deviation to the left.
↵cT cells administered at secondary site of recurrence.
↵dSurvival after second biopsy/relapse detected on day 64 as related to timeline in Figure S3D was 11.8 months.
Supplementary Data
Files in this Data Supplement:
- Supplementary methods - Supplementary methods. These are more detailed methods than can be included in the body of the article.
- Supplementary Tables S1-3 - Supplementary Tables S1-3. Table S1. T cell Product Release Criteria. Table S2. Manufacturing Feasibility. Table S3. Characteristics of Patients Who Received Study Treatment.
- Supplementary Figure S1 - Supplementary Figure S1. Catheter position with respect to resection site.
- Supplementary Figure S2 - Supplementary Figure S2. Consort flow diagram.
- Supplementary Figure S3 - Supplementary Figure S3. Treatment regimens for each patient, with relapse designated day 0.
- Supplementary Figure S4 - Supplementary Figure S4. 18F-Fluorodeoxyglucose ( F-FDG)-CT/PET of subjects UPN031 and UPN033
- Supplementary Figure S5 - Supplementary Figure S5. T cell detection in tumor tissue from UPN031.
Volume 21, Issue 18
