Fig. 6. Differential interaction modes of mono- and bivalent compstatin analogs.
a Compstatin-based drugs may interact with circulating (i.e., C3, C3c) or surface-tethered targets (i.e., C3b, C3bBb, iC3b). Depending on their valency, avidity may occur. b ITC analysis of solution interactions between C3c and compstatin analogs Cp01, Cp05*, Cp05-PEG-Cp05, and Cp40 shows 1:1 binding for monovalent analogs (N = 1) whereas the data of the bivalent Cp05-PEG-Cp05 fits a 2:1 ratio (N = 0.5). The displayed curve is a representative of two independent experiments per compound, obtained by peak-to-peak integration of the processed thermogram, as a function of At/Mt (titrant-to-titrate molar ratio). The integrated signal (black) is fitted to a 1:1 binding model (red curve) for determination of affinity (KD) and molecular ratio (N). Error bars represent the uncertainty associated to the integral calculation of each peak in the binding isotherm (as determined by AFFINImeter). Cp05* contains the linker necessary for the conjugation to PEG-40kDa; its binding profile is comparable to that of Cp05 (Supplementary Fig. 10c). c SPR analysis between immobilized C3b and mono- and bivalent Cp05 analogs reveal a strong enhancement of surface binding for Cp05-PEG-Cp05 over monovalent Cp05. d Isoaffinity kinetic plot for surface interactions of compstatin analogs with immobilized C3b, demonstrating that the development steps from Cp01 to Cp40 were enabled by improving dissociation rates with little impact on association rates. In contrast, the improved stability of the C3b complex with Cp05-PEG-Cp05 is accompanied by a marked drop in the association rate. PEG polyethylene glycol, RU resonance units.