Glioblastoma multiforme (GBM) is a highly malignant, non-curative tumor of the primary central nervous system with dismal prognosis. Treatment options are limited and conventional therapies, including surgery, radiotherapy, chemotherapy have not resulted in major improvements in the survival of patients. The advent of chimeric antigen receptor (CAR) T cell technology has opened new perspectives in the fight against cancer. Therefore, cell surface proteins that are suitable as targets for CAR T cells need to be identified. When considering a certain protein as a target for immunotherapy, two aspects have to be taken into account: Coverage of tumor cells as a factor for efficacy and the expression in healthy tissues under physiological conditions. The latter represents a potential threat, due to on-target/off-tumor toxicity. The novel MACSima™ Imaging Platform enables high-content, cyclic fluorescence-based imaging of individual biological samples in a fully automated fashion. We applied this method to subclassify different glioblastoma samples, to identify new glioblastoma marker and to analyze the toxicity profile of these markers. A selection of 96 markers, identified through flow cytometric analysis of cell surface expression on primary glioblastoma-derived xenografts, was used for characterization of diverse primary glioblastoma samples using the MACSima™ Imaging Platform. Cryosections were fixed using acetone, and each specimen was exposed to the 96 fluorochrome-conjugated antibodies by cycles of i) staining with antibody, ii) image acquisition, and iii) erasure of the fluorescence signal. Previously published markers, such as PDGFRα, p53, synaptophysin, CD44, nestin, podoplanin, GFAP, and EGFR were used to subclassify the glioblastoma into oligodendrocyte precursor (OPC), differentiated oligodendrocyte (DOC), astrocytic and mesenchymal (AsMes) or mixed subtype, according to Motomura et al.1 Analysis of well-established glioblastoma markers already used in CAR T cell-based clinical trials, such as IL-13Rα22, EGFRvIII3, or ErbB24 revealed a broad inter- and intratumor diversity of expression. Segmentation and correlation analysis enabled the selective detection of tumor cells and the identification of new glioblastoma specific markers, which might be candidates for future CAR T cell based therapies. To analyze the risk of “on-target/off-tumor” toxicity, differential expression of these markers on tumor tissue versus healthy tissue was determined. A higher expression level was detected on tumor tissue in comparison to healthy tissue in case of some candidates, underlining the capability of these markers for future CAR T cell-based therapies.

Citation Format: Sandy Reiß, Stefan Tomiuk, Jutta Kollet, Jan Drewes, Melanie Jungblut, Andreas Bosio. Comprehensive cyclic immunofluorescent analysis reveals new target candidates for CAR T cell based immunotherapy of glioblastoma multiforme [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6646.