Development of third generation anti-EGFRvIII chimeric T cells and EGFRvIII-expressing artificial antigen presenting cells for adoptive cell therapy for glioma.

Ayguen Sahin,Szofia Bullain,Carlos Sanchez,Ralph Weissleder,Peter Waterman,Bob S. Carter,Hiroshi Shiku

doi:10.1371/journal.pone.0199414

Ayguen Sahin, Szofia Bullain + Show 5 more

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https://doi.org/10.1371/journal.pone.0199414

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Abstract

Glioblastoma multiforme (GBM) is the most aggressive and deadly form of adult brain cancer. Despite of many attempts to identify potential therapies for this disease, including promising cancer immunotherapy approaches, it remains incurable. To address the need of improved persistence, expansion, and optimal antitumor activity of T-cells in the glioma milieu, we have developed an EGFRvIII-specific third generation (G3-EGFRvIII) chimeric antigen receptor (CAR) that expresses both co-stimulatory factors CD28 and OX40 (MR1-CD8TM-CD28-OX40-CD3ζ). To enhance ex vivo target specific activation and optimize T-cell culturing conditions, we generated artificial antigen presenting cell lines (aAPC) expressing the extracellular and transmembrane domain of EGFRvIII (EGFRVIIIΔ654) with costimulatory molecules including CD32, CD80 and 4-1BBL (EGFRVIIIΔ654 aAPC and CD32-80-137L-EGFRVIIIΔ654 aAPC). We demonstrate that the highest cell growth was achieved when G3-EGFRvIII CAR T-cells were cocultured with both co-stimulatory aAPCs and with exposure to EGFRvIII (CD32-80-137L-EGFRVIIIΔ654 aAPCs) in culturing periods of three to six weeks. G3-EGFRvIII CAR T-cells showed an increased level of IFN-γ when cocultured with CD32-80-137L-EGFRVIIIΔ654 aAPCs. Evaluation of G3-EGFRvIII CAR T-cells in an orthotropic human glioma xenograft model demonstrated a prolonged survival of G3-EGFRvIII CAR treated mice compared to control mice. Importantly, we observed survival of G3-EGFRvIII CAR T-cells within the tumor as long as 90 days after implantation in low-dose and single administration, accompanied by a marked tumor stroma demolition. These findings suggest that G3-EGFRvIII CAR cocultured with CD32-80-137L-EGFRVIIIΔ654 aAPCs warrants itself as a potential anti-tumor therapy strategy for glioblastoma.

Highlights

Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and aggressive malignant primary brain tumor in adults
To assess the best culture conditions for short-term and long-term propagation of this third generation EGFRvIII chimeric antigen receptor (CAR) approach and to test whether its antigen-specific activity can be enhanced, we developed artificial antigen presenting cell lines (EGFRVIIIΔ654 aAPC and CD32-80-137L-EGFRVIIIΔ654 aAPC), that express EGFRvIII on its cell surface
The expression of costimulatory molecules were determined by triple antibody staining and flow cytometric analysis, resulted in 99.8% for CD80, and 93.5% for CD137L in CD32-80-137L aAPC cells, and 99.6% for CD80, and 97.3% for CD137L in CD32-80-137L-EGFRVIIIΔ654 aAPC cells

Summary

Introduction

Glioblastoma multiforme (GBM) or grade IV astrocytoma is the most common and aggressive malignant primary brain tumor in adults. We and others have previously shown the efficacy of T-cell adoptive immunotherapy for glioblastoma using the CAR (chimeric antigen receptor) technology in preclinical models [1,2,3,4,5], and its safe application is currently being tested clinical studies [6]. We report here that assessments of both in vitro cytolysis of EGFRvIII target tumor cells as well as improved survival in an EGFRvIII positive intracranial human glioblastoma xenograft mouse model provide encouraging data that shows ‘third generation’ EGFRvIII-specific CAR T-cells, cocultured with EGFRvIII-specific aAPCs that express CD32, CD80, and CD137L (4-1BBL) co-stimulatory molecules presents itself to be an effective strategy for preparation of EGFRvIII directed CAR therapy for human glioma

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