Abstract 2807: Activated NK cells efficiently kill Glioblastoma through activating receptor-ligand interactions
Authors: Kmiecik Justyna , Navarro Andrea Gras , Enger Per Øyvind , Leiss Lina , Zimmer Jacques , Chekenya Martha 
Published: May 2015

Abstract
Glioblastoma (GBM) is the most malignant brain tumor, where the patients' survival remains dismally low despite multimodal therapies. Due to the immune suppressed status of GBM patients, adoptive cellular immunotherapy might represent an attractive therapeutic strategy. We hypothesized that allogeneic NK cells may be potent effectors against GBM due to their direct cytotoxicity and immunomodulatory properties through cytokine production. Nevertheless, it is not known how concurrent inhibitory and activating signals coordinate to regulate NK effector function against solid tumors. In vitro cytotoxicity assays using n=3 patient-derived GBM and K562 cells co-cultured with NK cells obtained from n=9 healthy donors revealed that activated NK cells efficiently killed GBM cells in a dose- and donor-dependent manner. The GBM cells variably expressed MHC class I, but highly expressed ligands for NKG2D receptor: MICA, ULBPs 1,2,3,5,6, and to a less extent MICB by flow cytometric phenotyping. Activated NK cells highly expressed NKp46, NKG2D, and NKG2A on the protein level. Analysis of high resolution genotyping of NK cell Killer Immunoglobulin-like Receptors (KIR) and HLA ligands expressed by GBM revealed that NK cell cytotoxic potency was associated with activating KIRs and NKG2D, and was partially independent of inhibitory KIRs repertoire in vitro. Blocking NKG2D receptor with specific antibody attenuated tumor lysis providing proof of concept for the contribution of this receptor. Experiments with antibodies blocking KIR2DS2 and KIR2DS4 receptors are in progress. Efficacy of NK cells obtained from donors with different KIR repertoires and infused intratumorally into human GBM-bearing NOD-SCID mice was investigated. The primary end points were animals' survival and mechanisms of therapeutic efficacy. Donor A lacked activating KIR2DS2 and KIR2DS4 and had inhibitory KIR-HLA ligands mismatched in the human GBM xenografts. In contrast, Donor B possessed activating KIR2DS2 and partial deletion KIR2DS4, as well as, a full repertoire of inhibitory receptors. NK cells from both donors significantly prolonged animal survival after single dose treatment compared to vehicle controls (p=0.0005 and p=0.0001; donor A and B respectively). Despite diminished inhibitory KIR-HLA ligand mismatch, donor B's NK cells extended the median survival to 163 days compared to 117.5 days of donor A´s NK cells. Furthermore, donor B´s NK cells significantly increased tumor apoptosis compared to control (p=0.04) and tumors treated with donor A´s NK cells (P=0.0004). Donor B´s NK cells significantly diminished tumor proliferation (p=0.02) and angiogenesis (p=0.04) compared to controls. In conclusion, allogeneic NK cells may be potent effectors against GBM mediated by activating signals through receptor-ligand interactions. However, further studies are required to evaluate their toxicity and applicability to GBM management.
Citation Format: Justyna Kmiecik, Andrea Gras Navarro, Per Øyvind Enger, Lina Leiss, Jacques Zimmer, Martha Chekenya. Activated NK cells efficiently kill Glioblastoma through activating receptor-ligand interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2807. doi:10.1158/1538-7445.AM2014-2807