Abstract B70: Universal adapter CAR-engineered NK-92 cells target patient-derived glioblastoma cancer stem cells
Authors: Grote Stefan , Chan Chun-Ho , Baden Caroline , Huber Stephan M. , Eckert Franziska , Mittelstaet Joerg , Kaiser Andrew , Seitz Christian , Schlegel Patrick , Handgretinger Rupert , Schleicher Sabine 
Published: March 2020

Abstract
Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor in adults. The current standard-of-care treatment, which includes surgical resection, radiation, and chemotherapy, extends the survival of patients to merely 14.6 months. GBM is virtually incurable due to its heterogeneity, which is represented by tumor cells with different patterns of gene expression (proneural, neural, classical, and mesenchymal GBM), the presence of GBM cancer stem (like) cells (CSC), and immune evasion. Thus, new therapeutic strategies directed at eliminating GBM CSC in particular are urgently needed. Utilizing fresh patient material from surgical resections, GBM CSC were successfully enriched and cultivated in serum-free medium and subsequently differentiated by switching to medium containing FBS. The gene expression pattern of both, GBM CSC and differentiated cells, was characterized. Furthermore, a screening platform was tailored for flow-cytometric characterization of patient-specific antigen expression of all newly established glioblastoma cell lines. In this work we present a promising immunotherapeutic approach towards eradication of GBM CSC by combining the universal targeting and controllability of our recently developed adapter chimeric antigen receptor (AdCAR) system with the “off-the-shelf” properties of the continuously expandable and well-characterized NK cell line NK-92. The AdCAR system is based on the unique properties of a novel scFv targeting a “neo”-epitope-like structure derived from the endogenous vitamin biotin, which can be linked to monoclonal antibodies (mAb). Utilizing these biotinylated mAb as adapter molecules, the system allows precise quantitative (on-/off switch) as well as qualitative (change and combination of target antigen) regulation of immune cell function. AdCAR-transduced NK-92 cells demonstrated significant target cell lysis, which, importantly, is largely independent from activation of endogenously expressed NK receptors and the presence of their ligands on target cells but mainly mediated by AdCAR activation through binding to the respective tumor antigen. In the presence of the respective antigen, first and foremost biotinylated antibodies directed at CD9, CD146, CD276, and EGFR were capable of inducing significant AdCAR NK-92-mediated lysis of GBM cells after 2 hours. Interestingly, due to overexpression of various target antigens on GBM cancer stem cells, CSC lysis was noticeably higher as compared to AdCAR-mediated lysis of differentiated glioblastoma cells. In conclusion, we have successfully generated a CAR-modified NK cell line, AdCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens, allowing universal and tunable targeting of glioblastoma cells. Most importantly, AdCAR NK-92 cells provide a promising strategy to eradicate GBM cancer stem (like) cells.
Citation Format: Stefan Grote, Chun-Ho Chan, Caroline Baden, Stephan M. Huber, Franziska Eckert, Joerg Mittelstaet, Andrew Kaiser, Christian Seitz, Patrick Schlegel, Rupert Handgretinger, Sabine Schleicher. Universal adapter CAR-engineered NK-92 cells target patient-derived glioblastoma cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B70.