Fig. 1: Interaction of heme with therapeutic Abs.

a Structural formula of Fe-protoporphyrin IX (heme). b Interaction of the repertoire of monoclonal IgG1 therapeutic antibodies to immobilized heme (left panel) and FA (right panel). Each circle on the panels represents the average binding intensity of a given therapeutic Ab analyzed in duplicate. The binding intensity for each Ab is obtained by division of reactivity to cofactor-conjugated gelatine by the reactivity of Ab to gelatine alone. The dashed line represents the tenfold increased binding over binding to gelatine alone. The right panel depicts the correlation of the binding to FA versus the binding to heme. The red circles represent Abs that are currently in clinical trials. The blue circles indicated the clinically approved Abs. The Spearman correlation analyses ρ = 0.69, P < 0.0001—indicates a significant correlation between the two parameters. The dashed lines display the 95%, confidence band. c Concentration-dependent binding of Abs to immobilized heme. The selected ten Abs were identified as top binders in the high-throughput heme-binding assay (panel b). Increasing concentrations 0.09–200 nM of the selected Abs were incubated with a heme-coated surface or with the control surface. Nonlinear regression analyses were performed by GraphPad Prism v.6 software. Each circle on the panels represents the average binding intensity ±SD of a given therapeutic Ab analyzed in duplicate. d Real-time interaction profiles of binding of heme to immobilized Abs (figitumumab and fulranumab). The black line depicts the binding profiles obtained after injection of serial dilutions of hemin (1250–19.5 nM). The red lines depict the fits of data obtained by global analysis using the Langmuir kinetic model.