Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications

The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ~20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FcγRIIb, and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. Mass spectroscopy of hexa-Fc reveals high-mannose, low-sialic acid content, suggesting that interactions with these receptors are influenced by the mannose-containing Fc. Molecular dynamics (MD) simulations provides insight into the mechanisms of hexa-Fc interaction with these receptors and reveals an unexpected orientation of high-mannose glycans on the human Fc that provides greater accessibility to potential binding partners. Finally, we show that this biosynthetic nanoparticle can be engineered to enhance interactions with the human neonatal Fc receptor (FcRn) without loss of the oligomeric structure, a crucial modification for these molecules in therapy and/or vaccine strategies where a long plasma half-life is critical.

. Structural characterization of Hexa-Fc. (A) Shown are top and side views of the hexamer with the C309 and tailpiece cysteine (here, C366) residues as yellow van der Waals (vdW) spheres. The C309 residues in neighboring monomers are covalently linked to each other in this model. The tailpiece cysteines in randomly selected monomers are also joined together. The tailpieces are shown in brown, the Fc of hIgG1 in blue, the sugars that are not mannose in red and the mannose residues as green spheres. (B) Tapping mode atomic force microscopy (AFM) images of hexa-Fc show birdcage structures of a size and shape consistent with the modeled hexameric complexes. Scale bar: 500nm, main figure; 60nm inset. (C) Superimposition of IgG1 b12 (pdb 1HZH) (grey) onto hexa-Fc (faded blue).

Figure S2. Hexa-Fc binds to CD19 + human B cells and CD14 + monocytes.
Characteristic FACs plot showing different populations of human PBMCs represented by forward (FSC) and side (SSC) scatter profiles (left panel). (A) Individual CD19 + B cells stained with anti-human CD19-FITC (shown by green dots) were gated (middle panel). Binding of 50µg of hexa-Fc (green trace) to gated human CD19 + B lymphocytes was detected using phycoerythrin (PE)-labeled goat (Fab' 2 ) anti-human IgG (right panel). (B) Individual CD14 + monocytes stained with anti-human CD14-APC-Cy7 (green dots for high CD14 expression and blue dots for low CD14 expression) were gated (middle panel). Binding of 50 µg of hexa-Fc (green trace for CD14 + high, blue trace for CD14 + low) to gated human CD14+ monocytes was detected using phycoerythrin (PE)-labeled goat (Fab' 2 ) anti-human IgG (right panels). Background binding detected with the PE-labeled goat (Fab' 2 ) anti-human IgG in the absence of hexa-Fc (grey traces). Data are representative of three separate independent experiments. Figure S3. Hexa-Fc preferentially binds FcγRIIb over FcRL5. FcRL4/CD32 (top panel) or FcRL5/CD32 (bottom panel) double-transfectants were pre-incubated with anti-FcRL5 blocking mAb 509F6 (orange trace) or anti-FcRL4 blocking mAb 413D12 (green trace). Anti-FcRL5 blocking antibody did not reduce binding of hexa-Fc but markedly reduced binding of heat-aggregated IgG, showing that hexa-Fc prefers to bind FcγRIIb when given the choice of either receptor. Red and blue traces show binding by 509F6 and 413D12 respectively in the absence of human Igs. Cell surface expression of FcRL5 and FcRL4 were confirmed using FITC-conjugated anti-FLAG M2 mAb. The FcγRIIb construct was not FLAG tagged. We therefore confirmed they were FcγRIIb positive with an anti-FcγRIIb antibody and/or isotype matched controls (methods). Data are representative of duplicate experiments.    Figure S8. Microtiter wells (Nunc) were coated with DC-SIGN at 10µg/ml in carbonate buffer pH9 and incubated over night at 4°C prior to blocking for 2h at room temperature (RT) in TSM (20mM Tris-HCl, 150mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 , 5% BSA) buffer pH 7.4. The wells were washed four times with TSM before addition of 100µl digested or undigested antibodies at 10µg/ml in TBS buffer to duplicate wells. After overnight incubation at 4 o C and washing as above, alkaline phosphataseconjugated anti-human IgG or IgM (1∶1000; Sigma) was added and incubated for 2h at RT. Wells were washed as above, and 100 µl of the substrate p-nitrophenyl phosphate (Sigma) added to each well and the absorbance measured at 405nm.  Figure S11. Structural details of the glycans within the Fc domain after ~125ns. The glycans in this structure, and most frequently throughout the trajectory, interact via their α1,3 branch mannose and central β mannose residues. While the α1,6 branch mannose residues in the glycan structure on the left are buried within the cavity, those on the right are clearly located at the cavity entrance. The sugars and protein are coloured as in Figure S1. Figure S12. Evaluation of the accessibility of the α1,6 branch mannose residues for the DC-SIGN CRD. The CRD is positioned so that the mannose residues in the crystal structure overlap those in the Fc-glycan. The Fc-glycan is coloured as in Figure S11, the CRD is pink, and the mannose glycan in the CRD crystal structure is grey. The van der Waals representation of the structure (right) more closely reflects the physical structure of the complex.
Trajectory of the Fc/FcRL5 complex during equilibration simulations. The time between adjacent frames is ~0.4ns and this movie reflects a moving average over 3 frames of the original trajectory to enable a clearer appreciation of the relative domain movements. Coloring scheme as in Figure 6.
Trajectory of the Fc/FcγRIII complex. The time between adjacent frames is ~0.4ns and this movie reflects a moving average over 3 frames of the original trajectory. Coloring scheme as in Figure S10.
Trajectory of the glycosylated Fc. The time between adjacent frames is ~0.1ns and this movie reflects a moving average over 3 frames of the original trajectory. The Fc is cyan, the mannose residues are green, and the non-mannose residues are red. Colored orange are the protein residues that are within 3Å of the sugar residues in each frame (roughly hydrogen-binding distance). The glycan in the foreground frequently adopts a configuration in which the mannose residues are near the entrance of the Fc cavity.

Supplementary Movie 4.
Trajectory of the glycosylated Fc. Similar to supplementary movie 3, but with the molecule rotated by 90⁰ about its long axis.