Figure 6

In vivo effects of hexameric-Fc. (A) ¹²⁵I γ1 Hexameric-Fc was administered to mice at 0.5 mg/kg, 2 mg/kg or 10 mg/kg. At indicated timepoints, plasma was collected from three mice per timepoint and concentration of protein bound radioactivity in plasma determined by direct measurement in a gamma counter. Values were corrected to calculate μg-equivalents of hexameric-Fc per mL of plasma. (B) γ4eng F234L F296Y hexameric-Fc administered to cynomolgus monkeys by IV bolus at 1 dose of 2 mg/kg. Concentrations of hexameric-Fc and smaller and larger human Fc-containing moieties were detected in plasma by mass spectroscopy. n of 3 animals, ± SEM. (C) Binding of γ1-hexameric-Fc to immobilised recombinant FcRn was investigated by SPR. Hexameric-Fc was titrated in a two-fold dilution series from 2.5μM to 39 nM. (D) To assess hexameric-Fc mediated FcγR blockade in cynomolgus monkeys, whole blood samples were collected after a 30 mg/kg IV dose of γ4eng F234L F296Y hexameric-Fc. Surface labelling of samples was carried out to identify monocytes (CD14⁺) and occupancy of FcγRs assessed using a AF647-conjugated γ4eng F234L F296Y prior to analysis by flow-cytometry. 3 animals, ± SEM. (E) To assess the effect of hexameric-Fc in an ITP model, 10 mg/kg hexameric-Fc was administered to mice IV, at the timepoints indicated, prior to addition of anti-CD41 (MWReg30) to induce platelet depletion. Whole blood samples were taken immediately prior to and 24 hour post anti-CD41 in order to determine platelet numbers. n = 6 mice per group, graph shows mean ± SEM, *=p < 0.05, ***=p < 0.01, by ANOVA and Dunnetts multiple comparison test.