Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: Characterisation of hexameric-Fc binding. ( A ) The binding of fluorescently tagged γ1 hexameric-Fc and IVIg to PBMC subsets was analysed by flow cytometry. Shown is 1 representative donor out of 3 independent donors. ( B ) Flow cytometric analysis of FcγR expression on PBMCs. Cells from the same donor as in ( A ) were stained with FcγR antibodies to analyse FcγRI, FcγRII and FcγRIII expression on PBMC subsets. ( C ) Binding of hexameric-Fc to HEK293 cells transfected with indicated FcγR constructs. Data shows the mean of 3 independent experiments + /- SEM. ( D ) Representative SPR traces showing the binding of γ4-hexameric-Fc and IgG4 to FcγRIIa. Hexameric-Fc was titrated in a two-fold dilution series from 1μM to 7.8 nM. IgG4 was titrated in a two-fold dilution series between 50μM and 0.39 μM.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: Binding Assay, Flow Cytometry, Expressing, Staining, Transfection, Construct

Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: In vitro disruption of FcγR function. ( A ) Macrophages were incubated with hexameric-Fc s or IvIg at 75 µg/ml for 1 hour. Cells were then washed and incubated for the indicated period. Cells were then labelled with fluorescently-conjugated hexameric-Fc. Data shows means from three donors ± SEM. ( B ) Pre-incubation with hexameric-Fc inhibits phagocytosis for 72hrs. Human monocyte-derived macrophages were incubated with the indicated hexameric-Fcs or IVIg at 75 µg/ml or 300 µg/ml for 1 hour, washed and cultured for 1 to 72 hours before co-culture with autologous CFSE-labelled B cell targets in the presence of 0.1 μg/ml anti-CD20. The disappearance of target cells was measured by flow cytometry after 18hrs and plotted as % inhibition of total antibody-dependent phagocytosis. Data are the mean ± SEM for three donors.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: In Vitro, Disruption, Incubation, Derivative Assay, Cell Culture, Co-Culture Assay, Flow Cytometry, Inhibition

Journal: Scientific Reports

Article Title: Multivalent Fc γ -receptor engagement by a hexameric Fc-fusion protein triggers Fc γ -receptor internalisation and modulation of Fc γ -receptor functions

doi: 10.1038/s41598-017-17255-8

Figure Lengend Snippet: In vivo effects of hexameric-Fc. ( A ) 125 I 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.

Article Snippet: Other assay conditions included TT only (1.0 μg/ml), or TT-ICs with hexameric-Fc (50 μg/ml) or FcγR blocking antibodies (R&D Systems, AF1330 anti-CD16 and AF1597anti-CD16, 20 μg/ml per antibody).

Techniques: In Vivo, Clinical Proteomics, Concentration Assay, Radioactivity, Mass Spectrometry, Binding Assay, Recombinant, Flow Cytometry, Comparison