Fig. 4
A, B Graphical representation of ADCC data. CA9hu-1 and CA9hu-2 antibody variant-dependent cell-mediated cytotoxicity. Comparison of CA IX-negative versus CA IX-positive target cell line samples, n = 2. C Fit curves determine EC50 of CA9hu-1(HC4LC4) antibody response using the GraphPad Prism® software. D Humanized antibody variants showed high ADCC activity in Reporter ADCC assay (Promega) tested on cells BT-20, JIMT-1, and 42MGBA naturally expressing CA IX in hypoxia. The signal of the control was set as 100%. E Humanized antibody variants showed high ADCC activity in comparison with irrelevant therapeutic antibody bevacizumab. This figure clearly shows that humanized antibody variants CA9hu-1 and CA9hu-2 retain the ability to activate the ADCC pathway and to mediate the cytotoxic effect on target cells expressing CA IX. Data in the graph represent the mean ± standard deviation values. Statistical significance of differences was assessed using Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001), n = 3