Targeting NKG2D ligands in glioblastoma with a bispecific T-cell engager is augmented with conventional therapy and enhances oncolytic virotherapy of glioma stem-like cells Journal for Immunotherapy of Cancer, 2024 Jan 01

"Patient-derived GSCs and primary GSCs can be targeted by NKG2D BiTE. Patient-derived glioma stem-like cells (GSCs), E57 cells, were cultured in DMEM supplemented with 10% fetal calf serum to promote differentiation or cultured in two different medias to promote GSC-like phenotype (EFL and AdMEM). (A) E57 cells were stained for total surface NKG2DLs using biotinylated <t>recombinant</t> human NKG2D-Fc and streptavidin-PE secondary. The figure shows representative flow cytometry histogram plots of E57 GSCs unstained (dotted line), isotype stained GSCs (gray dotted line), and stained with NKG2D-Fc-streptavidin-PE when cultured as differentiated cells (blue) or GSC-like cells (red). (B) Relative median fluorescent intensity (RMFI) of differentiated E57 cells and GSCs stained for total NKG2DLs compared with isotype. (C) E57 cells cultured in 10% DMEM, EFL or AdMEM were infected with G207-NKG2D BiTE (red) or G207-Control BiTE (blue) at a multiplicity of infection (MOI) of 0.1 or left uninfected (black) for 96 hours before assessing virus infectivity by GFP expression by flow cytometry. (D) E57 cells in each media were infected with G207-NKG2D BiTE (red circles) or G207-Control BiTE (blue circles) at an MOI of 0.1, or treated with 1 nM free NKG2D BiTE (red squares) or Control BiTE (blue squares) and co-cultured with PBMC-derived T cells at an effector:target ratio (E:T) of 5:1. After co-culturing for 96 hours, cells were harvested, stained and the percentage of CD25 + and (E) CD69 + T cells, and (F) E57 cell viability was assessed by flow cytometry. (G) Human GBM tissue obtained by cavitron ultrasonic surgical aspirator from one patient was dissociated and cultured in either neurobasal medium (NBM) supplemented with recombinant human <t>fibroblast</t> growth factor <t>(FGF)</t> and epidermal growth factor (EGF) to encourage a GSC phenotype, or in RPMI containing 10% FCS to encourage a differentiated phenotype. Primary GBM cells were infected with G207-NKG2D BiTE (red) or G207-Control BiTE (blue) at an MOI of 0.1 or left uninfected (black) for 96 hours before assessing virus infectivity by GFP expression by flow cytometry. (H) E57 cells in each media were infected with G207-NKG2D BiTE (red circles) or G207-Control BiTE (blue circles) at an MOI of 0.1, or treated with 1 nM free NKG2D BiTE (red squares) or Control BiTE (blue squares) and co-cultured with PBMC-derived T cells at an E:T ratio of 5:1. After co-culturing for 96 hours, cells were harvested, stained and the percentage of CD25 + and (I) CD69 + T cells was assessed by flow cytometry. Data represents the mean percentage of positive cells or RFMI from triplicate repeats±SD. Statistical significance was assessed by two-way ANOVA followed by Bonferroni post hoc analysis, or one way ANOVA followed by Tukey’s post hoc test for (B). Significance was assessed versus uninfected cells for (C) and (G) or untreated cells within the relevant cell type for (D) (E) (F) (H) and (I). (ns p>0.05, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001). AdMEM, advanced DMEM; ANOVA, analysis of variance; BiTE, bispecific T-cell engager; DMEM, Dulbecco’s Modified Eagle Medium; EFL, EGF, FGF, laminin; GBM, glioblastoma; GFP, green fluorescent protein; NKG2D, natural killer group 2 member D; NKG2DLs, NKG2D ligands; PBMC, peripheral blood mononuclear cell; RMFI, Relative median fluorescence intensity; RPMI, Roswell Park Memorial Institute. "