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Intracerebral delivery of a third generation EGFRvIII-specific chimeric antigen receptor is efficacious against human glioma

https://doi.org/10.1016/j.jocn.2013.03.012Get rights and content

Abstract

Chimeric antigen receptors (CAR)-transduced T cells hold great promise in the treatment of malignant disease. Here, we demonstrate that intracerebral injection with a human, epidermal growth factor receptor variant III (EGFRvIII)-specific, third generation CAR successfully treats glioma in mice. Importantly, these results endorse clinical translation of this CAR in patients with EGFRvIII-expressing brain tumors.

Introduction

Glioblastoma (GBM) is the most common and deadly primary malignant brain tumor, for which there are limited treatment options.1 Chimeric antigen receptors (CAR) represent a promising technology that redirects T cells to treat tumors via surface antibody-based domains translated in tandem with intracellular T cell signaling moieties. Costimulatory 4-1BB signalling has been shown to significantly improve the ability of CAR-tranduced T cells to persist and achieve antitumor T cell responses.2 However, such “third generation” CAR have not been tested for efficacy against intracerebral tumors. A mutation of the epidermal growth factor receptor, variant III (EGFRvIII), is frequently expressed on the surface of GBM but is completely absent from all normal tissues.3 Here, we demonstrate that an EGFRvIII-targeted, third generation CAR is specific and effective against human GBM cells in vitro and in vivo.

Section snippets

Cell lines

Human glioma cell lines U87MG and U87MG.ΔEGFR are previously described.4

EGFRvIII CAR retroviral vector and analysis

Human peripheral blood lymphocytes were transduced with EGFRvIII CAR as described.5 Cytokine staining for interferon γ (IFNγ) was performed according to manufacturer’s instructions (Cytofix/Cytoperm; BD Bioscience, San Jose, CA, USA).

In vivo experiments

Efficacy was tested in non-obese diabetic scid gamma mice. Glioma cells (5 × 104) and T cells were implanted intracerebrally as described.6

Statistical methods

Frequencies of IFNγ+ cells with respect to groups

Third generation EGFRvIII CAR is specific for EGFRvIII+ glioma

One limitation of potent CAR-based therapies has been lethal toxicity arising from affinity for antigens co-expressed on healthy tissues.7, 8 Targeting EGFRvIII, however, greatly reduces this risk of autoimmunity. Demonstrating its specificity, EGFRvIII CAR-transduced T cells did not exhibit detectable immunity to EGFRvIII-negative cells above untransduced levels. However, on incubation with U87MG.ΔEGFR, EGFRvIII CAR-transduced T cells yielded a significantly elevated frequency of

Discussion

Here, we have demonstrated that intracerebral injection of T cells expressing third generation EGFRvIII CAR can mediate safe, therapeutic responses against EGFRvIII-expressing tumors in the brain. Additionally, soluble peptide blockade was shown to specifically inhibit the functional activity of CAR, and may serve as a potential antidote for CAR targeting less tumor-specific targets.

Our third generation EGFRvIII CAR promises to foster potent T cell function beyond what would otherwise be

Conflicts of interest/disclosures

The authors declare that they have no financial or other conflicts of interest in relation to this research and its publication.

Acknowledgements

This work was supported by grants from the National Institutes of Health: SRC on Primary Tumors of the Central Nervous System P50 NS020023-28 and 3R01-CA-135272-02S1 supplement for Brain Tumor Stem Cell R01. Additional support was provided by 5P50-NS020023-29, 5R01-CA135272-02S1, 5R01-CA134844-03, 5UL1-RR024128-05, 5R21-NS067980-02, and 5R21-NS068057-02. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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