Structural characterization of GASDALIE Fc bound to the activating Fc receptor FcγRIIIa

Abstract

The Fc region of Immunoglobulin G (IgG) initiates inflammatory responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) through binding to activating Fc receptors (FcγRI, FcγRIIa, FcγRIIIa). These receptors are expressed on the surface of immune cells including macrophages, dendritic cells, and natural killer cells. An inhibitory receptor, FcγRIIb, is expressed on macrophages and other myeloid leukocytes simultaneously with the activating receptor FcγRIIa, thereby setting a threshold for cell activation. The affinity of IgG Fc for binding activating Fc receptors depends on IgG subclass and the composition of N-linked glycans attached to a conserved asparagine in the Fc CH2 domain. For example, Fc regions with afucosylated glycans bind more tightly to FcγRIIIa than fucosylated Fc, and afucosylated Fcs exhibit enhanced ADCC activity in vivo and in vitro. Enhanced pro-inflammatory responses have also been seen for Fc regions with amino acid substitutions. GASDALIE Fc is an Fc mutant (G236A/S239D/A330L/I332E) that exhibits a higher affinity for FcγRIIIa and increased effector functions in vivo compared to wild-type Fc. To explore its altered functions, we compared the affinities of GASDALIE and wild-type Fc for activating and inhibitory FcγRs. We also determined the crystal structure of GASDALIE Fc alone and bound to FcγRIIIa. The overall structure of GASDALIE Fc alone was similar to wild-type Fc structures, however, increased electrostatic interactions in the GASDALIE Fc:FcγRIIIa interface compared with other Fc:FcγR structures suggest a mechanism for the increased affinity of GASDALIE Fc for FcγRIIIa.

Keywords: Affinity; Fc receptors; Fc–FcR structure; GASDALIE Fc; IgG Fc; SDALIE Fc.

Figure 1

SPR binding assays of Fc:FcγR interactions. (a) Sensorgrams from conventional SPR experiments in which Fc proteins were injected over immobilized FcγRs. Experimental data (colored lines) were fit to a 1:1 binding model (black lines). Residual plots are shown below each set of sensorgrams. For most interactions, the association and/or dissociation phases of the sensorgrams do not fit a 1:1 binding model. In cases in which the association and dissociation rates are very fast, the 1:1 binding model appears to fit the sensorgrams, but the kinetic rate constants are outside of the detectable range of the instrument and the equilibrium RU values do not converge as the concentration of analyte is raised. (b) Competition SPR results. Equilibrium binding curves for interactions of the Fc variants (wtFc, SDALIE Fc, and GASDALIE Fc) with Fcγ receptors (FcγRIIIa, FcγRIIa, and FcγRIIb). Each curve represents an equilibrium binding experiment in which the free Fc concentration (y-axis) is plotted versus the competitor (FcγR) concentration (logarithmic x-axis). Data points (triangles, circles, or squares) were fit by non-linear regression to the second order root function to an equilibrium binding model (solid black line) as described in the Methods. The more a curve is shifted to the left, the stronger the Fc:FcγR binding (i.e., the higher the affinity).

Figure 2

Structure of GASDALIE Fc:FcγRIIIa_F158 complex. (a) Overview of the GASDALIE Fc:FcγRIIIa_F158 complex. FcγRIIIa_F158 is in pink ribbon representation and GASDALIE Fc is in blue ribbon representation with the G236/S239D/A330L/I332E mutations highlighted as red spheres and N-linked glycans shown as blue sticks. (b–c) Comparison of interfaces in the GASDALIE Fc:FcγRIIIa_F158 and wtFc:FcγRIIIa structures on Fc chain A (panel b) and Fc chain B (panel c). Residues involved in interactions are shown in magenta (FcγRIIIa) and blue (Fc) with oxygen atoms in red and nitrogens in blue. Dotted lines between atoms represent lectrostatic interactions (red) or hydrogen bonds (yellow). Residues that do not participate in hydrogen bonds or electrostatic interactions are shown in gray.

Figure 3

Comparison of the interfaces of FcγRIIa and FcγRIIb with GASDALIE Fc and wtFc using homology modeling. (a) Alignment of the D2 domains of FcγRIIa (PDB 3RY4, yellow), FcγRIIb (PDB 2FCB, green), and FcγRIIIa (pink; from the GASDALIE Fc:FcγRIIIa complex structure). (b) Sequence alignment of the D1 and D2 domains of FcγRIIa, FcγRIIb and FcγRIIIa. Identical residues are highlighted in green and similar residues are highlighted in gray. (c–d) Homology modeled interfaces of GASDALIE Fc and wtFc with FcγRIIa and FcγRIIb. Predicted electrostatic interactions (red) and hydrogen bonds (yellow) are shown as dashes. Residues that are not predicted to participate in the Fc:FcgR interfaces are shown in gray. (c) Interactions of chain A of GASDALIE Fc (left) and wtFc (right) with FcγRIIa (top) and FcγRIIb (bottom). (d) Interactions of chain B of GASDALIE Fc (left) and wtFc (right) with FcγRIIa (top) and FcγRIIb (bottom).

Figure 4

Structure of unbound GASDALIE Fc. (a) Comparison of structures of unbound GASDALIE Fc (cyan), SDALIE Fc (blue; PDB 2QL1), and wtFc (gray; PDB IDs: 3DO3, 2DTS, 3AVE, 4Q7D, 1HZH) after alignment of C_H3 domains. (b) Comparison of unbound GASDALIE Fc (cyan), FcγRIIIa-bound GASDALIE Fc (blue-gray), and SDALIE Fc (blue) after alignment of C_H3 domains. (c) wtFc structure (PDB: 3AVE) showing location of Cα atoms for Pro238, Phe241, Arg301, and Pro329 (orange spheres) used for C_H2 domain separation distance measurements. C_H2 domain separations in individual Fc structures in Table 2 were evaluated by measuring distances (dotted lines) between the corresponding orange spheres on each chain. Cα atoms for Asn297 (site of N-glycan attachment) indicated as blue spheres.