EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma
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Ali C. Ravanpay1,2,*, Juliane Gust3,4,*, Adam J. Johnson1, Lisa S. Rolczynski1, Michelle Cecchini2, Cindy A. Chang1, Virginia J. Hoglund1, Rithun Mukherjee1, Nicholas A. Vitanza5,6, Rimas J. Orentas1,6 and Michael C. Jensen1,6,7
1 Ben Towne Center for Childhood Cancer Research, Seattle Children’s Research Institute, Seattle, WA, U.S.A
2 University of Washington, Department of Neurological Surgery, Seattle, WA, U.S.A
3 University of Washington, Department of Neurology, Seattle, WA, U.S.A
4 Center for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA, U.S.A
5 Center for Clinical and Translational Research, Seattle Children’s Research Institute, Seattle, WA, U.S.A
6 University of Washington, Department of Pediatrics, Seattle, WA, U.S.A
7 University of Washington, Department of Bioengineering, Seattle, WA, U.S.A
* These authors contributed equally to this work
Keywords: glioblastoma; CAR T cell; EGFR; immunotherapy; mAb806
Received: August 14, 2019 Accepted: December 02, 2019 Published: December 17, 2019
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.
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