Fig. 5.

Absence of bystander cytotoxicity and translation into solid tumor model. a Intracellular caspase-3 activation as a measure of apoptosis in HLA-A2/CD45 dual-positive THP-1 (upper panels) and CD45-positive but HLA-A2-negative bystander cells (bottom panels) was assessed by flow cytometry in vitro after co-culture with donor PBMCs and CD45 or HLA-A2 targeting hemibodies as indicated (3 nM). The HLA-A2 specific BiTE was used as a positive control (left panel); data represent one out of two experiments that yielded similar results. b Luciferase-expressing dual antigen-positive THP-1 and single antigen-positive RAJI cells were injected subcutaneously in the left and right thigh, respectively. Donor T cells were injected i.v. at day 1 and saline (PBS), paired hemibodies and the BiTE control (8 µg/mouse) were administered s.c. daily until day 7. Data are representative for two independent experiments. c Immune deficient mice were challenged i.v. with luciferase-expressing human breast cancer cells MDA-MB-231, which co-express EGFR and Her2/neu antigens. After engraftment of lung metastases on day 3, PBMCs from a healthy donor were administered i.v. followed by s.c. injection of a buffer control (saline), a CD3 × EGFR BiTE or the combination of EGFR and Her2/neu-specific hemibodies. Tumor burden was visualized by IVIS Lumina XR Real-Time Bioluminescence Imaging on days 1 and 8. One in vivo experiment out of two is shown. For in vitro analyses, data represent means (±SD) of triplicate wells from at least two independent experiments, E:T = 10:1.