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. 2017 Jan 17;9(3):506–520. doi: 10.1080/19420862.2016.1274844

Figure 2.

Figure 2.

Affinity of different GpL-18D1-IgG1 fusion proteins for Fn14. (A) HT1080 and HT1080-Fn14-KO cells were analyzed by FACS with respect to Fn14 expression. (B) HT1080 and HT1080-Fn14-KO cells were seeded in the upper and lower half of a 24-well tissue culture plate. The next day, wells containing the 2 cell types were pairwise incubated (1.5 h, 37°C) with the indicated concentrations of the various GpL-18D1-IgG1 fusion proteins. After removal of the unbound GpL-18D1-IgG1 fusion protein molecules, cell-associated luciferase activity was measured by help of the Gaussia Luciferase Assay Kit. Specific binding values for the various GpL-18D1-IgG1 fusion proteins were calculated by subtracting the non-specific binding values derived of the HT1080-Fn14-KO cells from the Fn14+ parental HT1080 cells. KD values were obtained using the “nonlinear regression to a one-site specific binding curve” function of the GraphPad Prism5 software. Shown is one representative experiment for each fusion protein. (C) Determination of the KD values of the parental Fn14-specific IgG1 antibody 18D1 by heterologous competition binding experiments. The indicated concentrations of 18D1 were mixed with GpL(CT-HC)-18D1-IgG1 (250 ng/ml), GpL(CT-LC)-18D1-IgG1 (100 ng/ml) and GpL(NT-LC)-18D1-IgG1 (250 ng/ml). HT1080 cells were then incubated with these mixtures for 1.5 h at 37 °C. After removal of the unbound antibody fusion proteins, cell-associated luciferase activity was again determined with the Gaussia Luciferase Assay Kit. The binding data obtained were fitted with the “nonlinear regression to a one-site competitive binding curve” function of the GraphPad Prism5 software to calculate the Ki value of 18D1. Shown is one representative experiment for each GpL-18D1-IgG1 fusion protein. Average values and statistics of all experiments are summarized in Table 2. RLU, relative light units. (D) Concentration-dependent specific binding of GpL(CT-LC)-18D1-IgG1 to HT1080 cells was determined as in “B.” Specific binding values were normalized against the maximal binding (Bmax) value obtained by nonlinear regression to a one-site specific binding curve. Specific binding values were furthermore converted into the number of occupied Fn14 molecules per cell. (E) Concentration-dependent specific binding of GpL(CT-LC)-18D1-IgG1 and citrine(CT-LC)-18D1-IgG1 to HT1080 cells was determined as in “B.” Specific binding values averaged from 3 independent experiments are shown as fold over background. (F) Equilibrium binding studies with 3 fully independent batches of GpL(CT-LC)-18D1-IgG1 were repeatedly (n = 6) performed as described in “B.” n.s., no significant difference.