Fig. 4
From: Lymphocyte activation gene 3 is increased and affects cytokine production in rheumatoid arthritis
Surface plasmon resonance (SPR) results showing LAG-3, anti-LAG3 mAb, and Gal-3 binding kinetics. A SPR plot showing Gal-3 binding to LAG-3 in a dose-dependent way. B Fits to the experimental data were made with EVILFIT for the LAG-3/Gal-3 interaction. The distribution in ligand binding kinetics is shown in the two-dimensional grids. Each grid point represents a 1:1 interaction typified by KD and kd. A third coordinate, indicated with a color scale, represents the abundance of these interactions. C SPR plot and corresponding EVILFIT curve (D) showing Gal-3 binding to the antagonistic LAG-3 Ab (17B4) without the presence of LAG-3. Kinetics in a similar range as Gal-3/ LAG-3 interaction is seen. E SPR plot showing how Gal-3 interferes with the LAG-3/antagonistic LAG3 antibody binding in a dose-dependent way. Chip with immobilized antagonistic LAG-3-Ab (17B4). Flow containing a constant Fc:LAG3 amount and increasing concentrations of human recombinant Gal-3 were added. F The interaction between LAG-3 and LAG-3 mAb as a function of Gal-3 concentration is plotted with two-dimensional fits. To quantify the development in the distribution of binding kinetics, three bins (Bin 1, Bin 2, and Bin 3) were defined, guided by the major types of interactions observed for LAG-3, Gal-3, and LAG-3 mAb. G From bin 1, we see a typical signal from a mAb interaction (KD at 1 nM) at the lowest concentrations of Gal-3. This signal decreases with increasing Gal-3 concentrations and is lost above 1600 nM Gal-3. Bin 2 shows an intermediate state with complexes between LAG-3, Gal-3, and mAb that occurs with Gal-3 concentrations between 100 and 3200 nM Gal-3 (KD 10 µM). Bin 3 shows direct binding between Gal-3 and anti-LAG-3-mAb (KD 1 mM–1 µM)