Fig. 4

Effects of the BFP on target cell lysis and effector cell proliferation. a-e PBMC of healthy donors were incubated with leukemia cells treated with the indicated constructs (all 10 μg/mL) or left untreated. a Lysis of NB-4 leukemia cells was determined by 2 h cytotoxicity assays. Exemplary results (left panel) and combined data obtained with PBMC of 6 different healthy donors at an E:T ratio of 20:1 (right panel) are shown (Mean ± SEM). b, c Lysis of NB-4 leukemia cells was determined by flow cytometry based lysis assays at an E:T ratio of 20:1 after (b) 8 h and (c) 48 h using PBMC of 6 different donors (Mean ± SEM). d, e Cell death of THP-1 leukemia cells (E:T ratio 20:1) labelled with a red cell permeable dye was determined with an IncuCyte live cell imaging system using a Cytotox green cell death reagent over 96 h. Representative pictures at 0 h, 24 h and 72 h are shown (d) (THP-1 cells, red; dying cells, green; merged cells, yellow; magnification 4×). f Therapeutic efficacy of the two BFP was comparatively analyzed as follows: “% lysis NKG2D-CD16” – “% lysis NKG2D-CD3”. The net effect of the superior construct at the indicated time points is depicted. g PBMC of healthy donors were incubated with the indicated concentrations of the BFP in the presence of NKG2DL positive MOLM-14 cells for 72 h (E:T ratio 4:1). T cell proliferation was assessed by thymidine-uptake assays (left panel). Combined data obtained with PBMC of 4 different healthy donors at a concentration of 10 nM and with the parental CD3 mAb (UCHT1, 1.3 nM) as control (right panel) (Mean ± SEM)