A novel TanCAR targeting IL13Rα2 and EphA2 for enhanced glioblastoma therapy

Abstract

Chimeric antigen receptor T cell (CAR-T) therapy has been shown to be an effective strategy for combatting non-solid tumors; however, CAR-T therapy is still a challenge for solid tumors, such as glioblastoma. To improve CAR-T therapy for glioblastoma, a new TanCAR, comprising the tandem arrangement of IL13 (4MS) and EphA2 scFv, was generated and validated in vitro and in vivo. In vitro, the novel TanCAR-redirected T cells killed glioblastoma tumor cells by recognizing either IL-13 receptor α2 (IL13Rα2) or EphA2 alone or together upon simultaneous encounter of both targets, but did not kill normal cells bearing only the IL13Rα1/IL4Rα receptor. As further proof of principle, the novel TanCAR was tested in a subcutaneous glioma xenograft mouse model. The results indicated that the novel TanCAR-redirected T cells produced greater glioma tumor regression than single CAR-T cells. Thus, the novel TanCAR-redirected T cells kill gliomas more efficiently and selectively than a single IL13 CAR or EphA2 scFv CAR, with the potential for preventing antigen escape and reduced off-target cytotoxicity.

Keywords: CAR; CAR-T; EphA2; IL13; TanCAR; glioblastoma.

Graphical abstract

Figure 1

Functional characterization of the novel IL13 CAR in Jurkat T cells (A) Illustration of a cell-based luciferase reporter system for analyzing the biological activity of IL13 CAR. When a CAR expressed on the surface of Jurkat T cells (named the effector cell) carrying a luciferase reporter driven by an NFAT-response element was activated by its partner molecule present on the target cell, the luciferase expression level increased. (B) The biological activity assay of different CARs with U87 or THP-1 target cells using a cell-based luciferase reporter system. (C) The biological activity assay of different CARs with IL13Rα2-or IL13Rα1-engineered K562 target cells using a cell-based luciferase reporter system. Shown are representative plots of three independent experiments performed in triplicate. Statistically significant differences are indicated: ∗p < 0.05; ∗∗p < 0.01. IL13 (2MS) stands for IL13 (E13K.R109K), while IL13 (4MS) stands for IL13 (E13K.R66D.S69D.R109K).

Figure 2

Functional characterization of the novel bispecific IL13 (4MS)-EphA2 scFv-TanCAR in Jurkat T cells (A) Cartoon of the novel bispecific IL13 (4MS)-EphA2 scFv-TanCAR. To construct this novel TanCAR, IL13 (4MS) was inserted into a third-generation CAR backbone, then EphA2 scFv was linked with IL13 (4MS) by a (GGGS)3 linker. (B) Illustration of a cell-based luciferase reporter system for analyzing the biological activity of the novel TanCAR. (C) The biological activity assay of IL13 (4MS)-EphA2 scFv-TanCAR with U87 cells using a cell-based luciferase reporter system. (D) The biological activity assay of IL13 (4MS) CAR, EphA2 scFv CAR or IL13 (4MS)-EphA2 scFv-TanCAR with U87 cells using a cell-based luciferase reporter system. (E) The biological activity assay of IL13 (4MS) CAR, EphA2 scFv CAR, or IL13 (4MS)-EphA2 scFv-TanCAR with EphA2-engineered K562 target cells, IL13Rα2-engineered K562 target cells, or EphA2-IL13Rα2-engineered K562 target cells individually using a cell-based luciferase reporter system. Shown are representative plots of at three independent experiments performed in triplicate. Statistically significant differences are indicated: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. TanCAR stands for IL13 (4MS)-EphA2 scFv-TanCAR.

Figure 3

Functional characterization of the novel bispecific IL13 (4MS)-EphA2 scFv-TanCAR in human primary CD8⁺ T cells (A) The detection of the CAR expression level on the surface of human primary CD8⁺ T cells by flow cytometric assay with anti-Flag antibody. (B) The detection of IL2 levels in the supernatants from different groups of CD8⁺ CAR-T cells co-cultured with U87 cells or THP-1 cells. (C) The detection of IFNγ levels in the supernatants from different groups of CD8⁺ CAR-T cells co-cultured with U87 cells or THP-1 cells. (D) The detection of IL2 levels in the supernatants from different groups of CD8+ CAR-T cells co-cultured with U87 cells. (E) The detection of IFNγ levels in the supernatants from different groups of CD8⁺ CAR-T cells co-cultured with U87 cells. (F) The measurement of the cytotoxic activity of different groups of CD8⁺ CAR-T cells for U87 cells by LDH level assay. (G and H) The measurement of the cytotoxic activity of different groups of CD8+ CAR-T cells for THP-1 cells by LDH level assay (H) The measurement of the cytotoxic activity of different groups of CD8+ CAR-T cells for U87 cells by LDH level assay. Shown are representative plots of three experiments performed in triplicate. Statistically significant differences are indicated: ∗p < 0.05; ∗∗p < 0.01. TanCAR stands for IL13 (4MS)-EphA2 scFv-TanCAR.

Figure 4

Analysis of the antitumor activity of IL13 (4MS)-EphA2 scFv-TanCAR CD8⁺ T cells in a glioblastoma animal model (A) The detection of tumor progression by bioluminescence imaging. (B) The growth curve of tumors from the different groups treated with CAR-T cells. The tumor volume data are represented as mean ± standard deviation (mm³). (C) Photographs of tumors from different groups treated with CAR-T cells. On day 28 after tumor inoculation, tumors were resected immediately after euthanasia, and photographs of three samples from each group are shown. (D) Quantification of tumors from different groups treated with CAR-T cells by tumor weight. The tumor weight data are represented as mean ± standard deviation (mg). Statistically significant differences are indicated: ∗∗p < 0.01. TanCAR stands for IL13 (4MS)-EphA2 scFv-TanCAR.

Figure 5

Analysis of the tumor cell proliferation and homing abilities of CAR-T cells by H&E and immunohistochemical staining of tumor tissue samples The immunohistochemical staining of tumor samples with anti-Ki67 and anti-human CD3 indicates the distribution of tumor cells and the human CD3⁺ T cells, respectively. TanCAR stands for IL13 (4MS)-EphA2 scFv-TanCAR.