EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma.

Ali C Ravanpay,Juliane Gust,Cindy A Chang,Rimas J Orentas,Virginia J Hoglund,Adam J Johnson,Michael C Jensen,Nicholas A Vitanza,Michelle Cecchini,Rithun Mukherjee,Lisa S Rolczynski

doi:10.18632/oncotarget.27389

Abstract

Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells.

Highlights

Solid tumor treatment with chimeric antigen receptor (CAR) T cells is currently limited by the paucity of cell surface targets that are tumor restricted, broadly expressed throughout the tumor, and integrally associated with tumorigenicity
Since the length of CAR extracellular spacer domains has been shown to affect CAR mediated cellular cytotoxicity [35], we addressed the functional impact of spacer lengths on EGFR806-CAR T cell activity by engineering CARs with modular spacer domains designated short (S, IgG4hinge alone), medium (M, IgG4hinge-CH3), and long (L, IgG4hinge-CH2-CH3) (Figure 1A)
We have developed a CAR based on the tumorspecific mAb806, which is selectively activated by tumor-expressed epidermal growth factor receptor (EGFR), be it full-length or the truncated EGFRvIII variant

Summary

Introduction

Solid tumor treatment with CAR T cells is currently limited by the paucity of cell surface targets that are tumor restricted, broadly expressed throughout the tumor, and integrally associated with tumorigenicity. EGFR amplification, overexpression, or mutation is present in approximately half of glioblastomas and other malignant CNS tumors, including ependymoma and medulloblastoma, in both children and adults [5,6,7,8,9,10,11,12]. EGFR-targeted interventions such as tyrosine kinase inhibitors have had limited success in CNS tumors, likely due to difficulty accessing the tumors and tumor evasion of these pathway-based drugs [13, 14]. CAR T cells targeting EGFRvIII, a truncation mutant of EGFR that is common in glioblastoma, were safe but unable to eradicate the tumor, likely due to nonuniform expression of the target protein and antigen escape [15, 16]

Methods

Results

Conclusion