Fig. 2

Vaccine-induced antibody (VIA) in serum from mice immunized with adenovirus encoding full length human epidermal growth factor receptor 3 (HER3-VIA) mediates multiple mechanisms of action against human breast tumor cell lines in vitro. a Complement dependent cytotoxicity was measured against human epidermal growth factor receptor 3 (HER3)-expressing (BT474, T47D, MDA-MB-468, BT474M1) and HER3-negative (MDA-MB-231) cell lines. Percentage cytolysis is reported: *p < 0.0001, **p < 0.001, ***p < 0.05. b Antibody-dependent cell-mediated cytotoxicity was measured against HER3-expressing JC-HER3 cells and parental JC cells (HER3-negative) using mFcγRIV antibody-dependent cellular cytotoxicity (ADCC) Reporter Bioassay. Target cells were pre-incubated with 1:10 dilution of HER3-VIA or green fluorescence protein (GFP)-VIA for 30 min at room temperature, and then effector cells were applied per manufacturer’s instructions. Two different effector-target ratios (3:1, 7.5:1) were tested. After 6 h of co-incubation, Bio-Glo™ Reagent was added, and luminescence was measured. Luminescence of JC cells was subtracted from luminescence of JC-HER3 cells for each VIA and is shown: *p < 0.0001. c Proliferation of HER3-expressing (BT474, T47D, MDA-MB-468, BT474M1) or HER3-negative cell line (MDA-MB-231) was measured in a 72-h 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay in response to HER3-VIA or control (LacZ-VIA or GFP-VIA): *p < 0.0001. d Effect of HER3-VIA on HER3 expression and signaling pathway was analyzed by western blotting. BT474M1 cells were incubated with HER3-VIA or GFP-VIA (1:100 dilution) in vitro at 37 °C for 3 h. GAPDH, glyceraldehyde-3-phosphate dehydrogenase