Fig. 1

Anti-Gal binds to AGI-134-treated human cancer cells and activates CDC and ADCC. a Human SW480 and A549 cancer cells were treated with PBS (open histograms) or the indicated concentrations of AGI-134 (grey and black histograms). The cells were then incubated with affinity purified human anti-Gal IgG or 25% heat-inactivated human serum. Anti-Gal antibody binding was detected with fluorescently-labeled secondary antibodies and samples analyzed by flow cytometry. Representative histogram overlays from two to three independently conducted experiments for each data set are shown. b SW480 and A549 cells were treated with half-log dilutions of AGI-134 and incubated with 50% normal (NHS) or heat-inactivated (iNHS) human serum. In some experiments, SW480 cells were exposed to C7 depleted serum ± 70 µg/mL C7. Cell viability was determined using a luminescence-based cell viability assay and data normalized and expressed as percentage viability. Representative data from 3 independent experiments are shown, with mean values ± SD. c A549 cells were treated with PBS or 0.5 mg/mL AGI-134 and then co-cultured with Promega’s ADCC reporter bioassay effector cells in a 25:1 effector:target cell ratio, in the presence or absence of 30 µg/mL affinity purified human anti-Gal IgG for 6 h. Induction of ADCC over no anti-Gal antibody controls was determined by addition of Bio-Glo Luciferase reagent to quantify reporter gene expression downstream of FcγRIIIa. For assessment of target cell killing by NK cells, CHO-K1 cells were treated with PBS or 1 mg/mL AGI-134 and pre-incubated with 30 µg/mL affinity purified human anti-Gal IgG, before co-culture with IL-2-activated human NK cells. After 4–6 h of co-culture the percentage of dead CHO-K1 cells was determined by incorporation of the viability dye 7-AAD into the target cells. Data shown is the mean + SEM for three (reporter bioassay) or six (cell killing assay) independent experiments