B7-H3 as a Novel CAR-T Therapeutic Target for Glioblastoma

Abstract

Glioblastoma (GBM) remains one of the most malignant primary tumors in adults, with a 5-year survival rate less than 10% because of lacking effective treatment. Here, we aimed to explore whether B7-H3 could serve as a novel therapeutic target for GBM in chimeric antigen receptor (CAR) T cell therapy. In this study, a CAR targeting B7-H3 was constructed and transduced into T cells by lentivirus. Antitumor effects of B7-H3-specific CAR-T cells were assessed with primary and GBM cell lines both in vitro and in vivo. Our results indicated that B7-H3 was positively stained in most of the clinical glioma samples, and its expression levels were correlated to the malignancy grade and poor survival in both low-grade glioma (LGG) and GBM patients. Specific antitumor functions of CAR-T cells were confirmed by cytotoxic and ELISA assay both in primary glioblastoma cells and GBM cell lines. In the orthotropic GBM models, the median survival of the CAR-T-cell-treated group was significantly longer than that of the control group. In conclusion, B7-H3 is frequently overexpressed in GBM patients and may serve as a therapeutic target in CAR-T therapy.

Keywords: B7-H3; chimeric antigen receptor; glioblastoma; immunotherapy; low grade glioma.

Figure 1

Analysis of the Expression Level of B7-H3 in Glioma Sample (A) Representative cases of 34 diagnosed glioma samples including para cancer tissues from each grade. (B and C) Differential expression of B7-H3 in normal brain tissue and glioma with different grade (B) or type (C) was analyzed with RNA-seq datasets from Oncomine. (D and E) Survival analysis of LGG (D) and GBM (E) patient was shown (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2

B7-H3 Expression Level in Primary GBM Cells and GBM Cell Lines (A and C) Immunofluorescence staining indicated the expression of B7-H3 (red) on cell surface in GBM primary cells (A) and glioma cell lines (C). (B and D) Consistent with the immunofluorescence result, the flow cytometry analysis shows that high-level expression of B7-H3 was observed in UPN1 among the primary GBM cells (B) and in A172 cells among the GBM cell lines (D).

Figure 3

Construction of B7-H3-Specific CAR-T Cells and Cytotoxic Assays (A) Lentiviral vector construct with the EF1α promoter followed by the leader sequence, anti-B7-H3 scFv, hinge, CD8 transmembrance domain, CD28, 4-1BB, CD3ζ, P2A, and mCherry. (B and C) The transduction efficiency of CAR was measured using fluorescence microscope (B) and flow cytometry analysis of mCherry (C). (D) NT and CAR-T cell subsets and phenotype were analyzed by flow cytometry at 10 days after the transduction of CAR lentivirus. (E) The B7-H3 expression level of lenti-transduced U87 cells was assessed by flow cytometry, and the positive rate was 100%. (F and G) ⁵¹Cr-release assays of CAR-T cells against B7-H3^+/− cell lines (F) and primary GBM cells (G) in different E:T ratios (*p < 0.05).

Figure 4

Tumor Recognition and Cytokine Production of B7-H3-Specific CAR-T Cells In Vitro (A) Images of co-culture of vehicle and CAR-T cells with autologous GBM cells or GBM cell lines in E:T ratios of 4:1 and 8:1 at 8 h. (B–E) The IFNγ (B: GBM cell lines; C: primary GBM cells) and IL-2 (D: GBM cell lines; E, primary GBM cells) secretion levels of vehicle and CAR-T cells co-cultured with target cells (60,000 T cells to 15,000 tumor cells) for 12 h were measured by ELISA kit (*p < 0.05).

Figure 5

Antitumor Efficacy of B7-H3-Specific CAR-T Cells In Vivo (A and E) The treatment scheme of orthotropic GBM PDX (A) and U87-B7-H3 xenograft model (E) including intracranial injection of tumor cells, vein-tail injection of vehicle and CAR-T cells, and the timing of in vivo imaging. (B and F) Images of bioluminescence signal in mouse brain before and after injection of vehicle and CAR-T cells in orthotropic GBM PDX (B) and U87-B7-H3 xenograft model (F). (C and G) Tumor total flux (p/s) was calculated by using Living Image software after tail-vein injection of vehicle and CAR-T cells in orthotropic GBM PDX (C) and U87-B7-H3 xenograft model (G). (D and H) Log rank test was used to analyze the overall survival of tumor-bearing mice (*p < 0.05) in orthotropic GBM PDX (D) and U87-B7-H3 xenograft model (H).