Safety and antitumor activity of GD2-Specific 4SCAR-T cells in patients with glioblastoma

Abstract

Background: This study aimed to validate whether infusion of GD2-specific fourth-generation safety-designed chimeric antigen receptor (4SCAR)-T cells is safe and whether CAR-T cells exert anti-glioblastoma (GBM) activity.

Methods: A total of eight patients with GD2-positive GBM were enrolled and infused with autologous GD2-specific 4SCAR-T cells, either through intravenous administration alone or intravenous combined with intracavitary administration.

Results: 4SCAR-T cells expanded for 1-3 weeks and persisted at a low frequency in peripheral blood. Of the eight evaluable patients, four showed a partial response for 3 to 24 months, three had progressive disease for 6 to 23 months, and one had stable disease for 4 months after infusion. For the entire cohort, the median overall survival was 10 months from the infusion. GD2 antigen loss and infiltrated T cells were observed in the tumor resected after infusion.

Conclusion: Both single and combined infusions of GD2-specific 4SCAR-T cells in targeting GBM were safe and well tolerated, with no severe adverse events. In addition, GD2-specific 4SCAR-T cells partially mediate antigen loss and activate immune responses in the tumor microenvironment. Validation of our findings in a larger prospective trial is warranted.

Trial registration: ClinicalTrials.gov Identifier: NCT03170141 . Registered 30 May 2017.

Keywords: 4SCAR-T; GBM; GD2; Safety; Tumor microenvironment.

Fig. 1

Protocol schema and consort flow diagram. A Protocol schema for GD2 testing, enrollment, generation of 4SCAR-T cells, lymphodepletion, 4SCAR-T cell infusion, and follow-up. B Consort diagram showing the number of patients assessed and enrolled on the study

Fig. 2

Design and killing assay of GD2 specific 4SCAR-T cells. A Design of GD2 specific 4SCAR-T cells. B Study design of killing assay of peripheral blood mononuclear cells (PBMCs), CD19 specific 4SCAR-T cells and GD2 specific 4SCAR-T cells against primary GBM cells. The primary GBM cells transduced with a lentiviral wasabi green fluorescence reporter gene were used as target cells, and plated into 48-well plate before co-culturing with different cells (E:T ratio 4:1). C Fluorescence microscopy of primary GBM cells from Patient 01 on 24, 48 and 72 h after co-culture. D-K The percentage of lysed primary GBM cells from indicated patients. All data are represented as the mean ± SEM. *p < 0.05, GD2 4SCAR-T vs. PBMC; #p < 0.05, GD2 4SCAR-T vs. CD19 4SCAR-T (One-Way ANOVA)

Fig. 3

In vivo persistence of GD2-specific 4SCAR-T cells in peripheral blood. The copy number of GD2-specific 4SCAR-T cells in peripheral blood of indicated patients was determined by RT-qPCR analysis

Fig. 4

MRI scan of brain and overall survival post GD2 specific 4SCAR-T cell infusion. A-H Magnetic resonance imaging (MRI) scan of the brain before and 4 weeks after GD2 specific 4SCAR-T cell infusion. I Swimmer’s plot describing disease status and overall survival for each patient. PD, progressive disease. SD, stable disease. PR, partial response. A, Alive. D, Dead

Fig. 5

Immune modulation after GD2 specific 4SCAR-T cell infusion. A Hematoxylin and eosin (H&E) staining, IHC staining for GD2, CD3, CD4, CD8 an CD68 in pre- and post-GD2 specific 4SCAR-T cell infusion specimens from Patient 01. B Multiplex immunofluorescence staining of GD2, CD8 and CD163 in pre- and post-GD2 specific 4SCAR-T cell infusion specimens from Patient 01. C Circulating levels of IL-6, TNF-α, and IFNγ in cerebrospinal fluid (CSF) from Patient 01. (D) Circulating levels of IL-6 and TNF-α in serum from Patient 01

Fig. 6

Cytokine modulation in peripheral blood after GD2 specific 4SCAR-T cell infusion. (A-G) Circulating levels of IL-6 and TNF-α in serum from indicated patients