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Abstract
The purpose of this study was to generate a panel of donor-derived primary cells, expand them ex-vivo in to sufficient numbers to utilize as targets for evaluating potential normal tissue toxicity of epidermal growth factor receptor mutation variant three (EGFRvIII)-specific chimeric antigen receptors (CAR) prior to use in clinical trials for patients with glioblastoma (GBM). Nine cell types were obtained, including different epithelial, endothelial, bone, smooth muscle, cardiac, neural, hematopoetic, stem cells, and keratinocytes. Cells were expanded with individual specialized media and protocols for between 8-12 passages. After expansion, primary cell identity was confirmed by morphology, ICC and IHC for characteristic markers. Levels of EGFR and EGFRvIII in each cell type were determined by qRT-PCR. To evaluate potential normal-cell toxicity, CAR T cells were co-cultured with each type of primary cell and function was evaluated in two ways: i) T cell activation was measured by staining and flow cytometry of CD3+ T cells stained intracellularly for CD107a, or GzmB, TNFalpha, IFNgamma, IL-2 cytokines. ii) Target cell lysis was evaluated by labeling primary cells with 51Cr prior to 4 hour co-culture with increasing numbers of CAR T cells, and measuring chromium-release. None of the primary cells showed expression of EGFRvIII, although several, in particular keratinocytes and renal epithelial, had high levels of EGFR. In functional assays, while the EGFR CAR T cells recognized and lysed EGFR expressing cell types, EGFRvIII CARs showed T cell activation and target lysis only of EGFRvIII expressing tumors. EGFRvIII CAR 2173 was selected for use in clinical trials at UPENN and UCSD, treating patients with GBM. To date, 6 patients have been infused with 2173 EGFRvIII CAR T cells, with no observed toxicity. All patients had detectable expansion of CAR T cells in vivo in blood, and one patient with subsequent tumor resection had detectable intra-tumoral CAR T cells. These CARs appear to be safe, persist in vivo and traffic into GBM tumor. An update on the clinical trial will be presented at the conference.
Note:This abstract was not presented at the conference.
Citation Format: Alexandria P. Cogdill, Alina Boesteanu, Chong Xu, Kathleen Haines, John Scholler, Joseph Fraietta, Yangbing Zhao, Xiaojun Liu, Jennifer Morrissette, Bruce Levine, Simon Lacey, Andreas Loew, Reshma Singh, Jennifer Brogdon, Donald M. O'Rourke, Marcela V. Maus, Carl H. June, Laura A. Johnson. Toxicity testing of EGFRvIII CAR-based immunotherapy of glioblastoma: From bench to bedside. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B139.