Fig. 3: Heme-induced antigen-binding polyreactivity of Abs.

a ELISA analysis of the binding of the repertoire of therapeutic Abs to immobilized human (FVIII, C3) and bacterial (LysM AtlA) proteins. Each circle depicts the average reactivity of an individual Ab obtained from duplicate samples. The right panels show the global reactivity profile of the repertoire of therapeutic Abs. The protein binding intensity was obtained by dividing the reactivity of given Ab after exposure to heme by the reactivity of the same Ab in its native form. The dashed line represents a twofold increase in the binding. The red circles represent Abs that are currently in clinical trials. The blue circles indicate the clinically approved Abs. b Correlation between reactivities of the therapeutic Abs to different protein antigens upon exposure to heme. Matrix depicts the correlation analyses between the reactivity of heme-treated Abs to different proteins and the average reactivity toward the three proteins. The correlation analysis was performed using Spearman’s rank-order test. The values of the correlation coefficients (ρ) are shown in the matrix. All P values are <0.005. The right panel illustrates the statistical significance (P values). c Immunoreactivity of selected Abs (top 10 heme-sensitive Abs) toward antigens present in the total lysate of B. anthracis. d The three-dimensional structure of tigatuzumab was modeled by sequence-based variable region modeling algorithm RosettaAntibody implemented on ROSIE web server (http://rosie.rosettacommons.org/). The putative heme-binding site was then predicted by docking of the protoporphyrin IX molecule to the variable region using the SwissDock web service protein–ligand docking platform-based on the EADock DSS algorithm (http://www.swissdock.ch/). The figure shows the side and top view of the variable region of tigatuzumab. Gray: variable domain of light chain; blue: variable domain of heavy chain. Heme is shown in red.