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. 2013 May 9:6:33.
doi: 10.1186/1756-8722-6-33.

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma

Chan-Juan Shen  1 , Yu-Xiu Yang, Ethan Q Han, Na Cao, Yun-Fei Wang, Yi Wang, Ying-Ying Zhao, Li-Ming Zhao, Jian Cui, Puja Gupta, Albert J Wong, Shuang-Yin Han
Affiliations

Chimeric antigen receptor containing ICOS signaling domain mediates specific and efficient antitumor effect of T cells against EGFRvIII expressing glioma

Chan-Juan Shen et al. J Hematol Oncol. .

Abstract

Background: Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells appears to be a promising immunotherapeutic strategy. CAR combines the specificity of antibody and cytotoxicity of cytotoxic T lymphocytes, enhancing T cells' ability to specifically target antigens and to effectively kill cancer cells. Recent efforts have been made to integrate the costimulatory signals in the CAR to improve the antitumor efficacy. Epidermal growth factor receptor variant III (EGFRvIII) is an attractive therapeutic target as it frequently expresses in glioma and many other types of cancers. Our current study aimed to investigate the specific and efficient antitumor effect of T cells modified with CAR containing inducible costimulator (ICOS) signaling domain.

Methods: A second generation of EGFRvIII/CAR was generated and it contained the EGFRvIII single chain variable fragment, ICOS signaling domain and CD3ζ chain. Lentiviral EGFRvIII/CAR was prepared and human CD3+ T cells were infected by lentivirus encoding EGFRvIII/CAR. The expression of EGFRvIII/CAR on CD3+ T cells was confirmed by flow cytometry and Western blot. The functions of EGFRvIII/CAR+ T cells were evaluated using in vitro and in vivo methods including cytotoxicity assay, cytokine release assay and xenograft tumor mouse model.

Results: Chimeric EGFRvIIIscFv-ICOS-CD3ζ (EGFRvIII/CAR) was constructed and lentiviral EGFRvIII/CAR were made to titer of 106 TU/ml. The transduction efficiency of lentiviral EGFRvIII/CAR on T cells reached around 70% and expression of EGFRvIII/CAR protein was verified by immunoblotting as a band of about 57 kDa. Four hour 51Cr release assays demonstrated specific and efficient cytotoxicity of EGFRvIII/CAR+ T cells against EGFRvIII expressing U87 cells. A robust increase in the IFN-γ secretion was detected in the co-culture supernatant of the EGFRvIII/CAR+ T cells and the EGFRvIII expressing U87 cells. Intravenous and intratumor injection of EGFRvIII/CAR+ T cells inhibited the in vivo growth of the EGFRvIII expressing glioma cells.

Conclusions: Our study demonstrates that the EGFRvIII/CAR-modified T cells can destroy glioma cells efficiently in an EGFRvIII specific manner and release IFN-γ in an antigen dependent manner. The specific recognition and effective killing activity of the EGFRvIII-directed T cells with ICOS signaling domain lays a foundation for us to employ such approach in future cancer treatment.

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Figures

Figure 1
Figure 1
Evaluation of EGFRvIII/CAR expression on CD3+ T cells. (A) Schematic representation of EGFRvIII/CAR. It consists of EGFRvIII scFv, the hinge and transmenbrane (TM) region of human CD8α, ICOS signaling domain, and human CD3ζ chain. IgG κ chain was used as signal peptide (SP). (B) Surface expression of EGFRvIII/CAR on CD3+ T cells. Left, isotype control; Middle, anti-human CD3-FITC and anti-human CD8-PE staining (BD Biosciences); Right, anti-mouse F(ab)2-FITC (eBiocience) and anti-human CD8-PE staining. (C) Immunoblot analysis of EGFRvIII/CAR expression. Lysates of untransduced T cells (lanes 1) and EGFRvIII/CAR transduced T cells (lane 2) were separated by SDS-PAGE under reducing condition. Goat anti-human CD3ζ antibody was used to detect the endogenous and chimeric CD3ζ expression.
Figure 2
Figure 2
Functional analysis of EGFRvIII/CAR+ T cells. (A) Cytotoxic activity of EGFRvIII/CAR+ T cells. (Left) Target cells were EGFRvIII-expressing U87 cells which can be lysed by EGFRvIII/CAR+ T cells, not by GFP+ and NT T cells. (Right) Target cells were EGFRvIII-negative U87 cells, which can not be lysed by either EGFRvIII/CAR+ or control T cells. (B) Cytokine release of EGFRvIII/CAR+ T cells. Only EGFRvIII/CAR+ T cells released significant amount of IFN-γ when co-cultured with EGFRvIII-expressing U87 cells. No increased IFN-γ expression was detected when co-cultured with EGFRvIII-negative U87 cells, nor from control GFP+ and NT T cells. Results are the mean and the SD from experiments in triplicate. * indicate P<0.05.
Figure 3
Figure 3
In vivo antitumor activity of EGFRvIII/CAR+ T cells. EGFRvIII expressing U87 cells were used for xenograft mouse model. EGFRvIII-bearing BALB/cA-nude mice received different treatments: group A, EGFRvIII/CAR+ T cells (IT); group B, EGFRvIII/CAR+ T cells (IV); group C, GFP+ T cells (IV); group D, PBS (IV). Results are expressed as a mean tumor volume (mm3±SD) with n = 5 for all groups. The standard deviation (SD) is represented by error bars.
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