Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of Intravenous Immunoglobulin G (IVIG) requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of IVIG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc CH2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully di-sialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the CH2 domain is associated with the switch from pro- to anti-inflammatory activity of the Fc.

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.

Antibodies (Ab) mediate pro-inflammatory effector functions through the engagement of Fc receptors (FcγRs) expressed on leukocytes. A complex, N-linked glycan attached to the Fc domain of IgG regulates the interaction to FcγRs by maintaining the Fc in a biologically active conformation. However, attaching sialic acid sugar residues to this Fc-associated glycan reduces affinity for FcγRs and confers binding to an alternate class of receptors, the C-type lectins (DC-SIGN). This switch in receptor specificity coincides with a switch in effector function in vivo as sialylated IgG (sFc) suppresses inflammation in mouse models of autoimmunity. The structural mechanism that regulates the sialic acid-dependent change in Ab effector function remains poorly understood. Biophysical analysis reveals that sialylation destabilizes the Fc fragment by increasing the hydrophobic surface area exposed to solvent. Upon solving a crystal structure of sFc, we observe that this loss of stability correlates with inter-domain flexibility such that the sFc in the crystal is found in both “open” and “closed” states. Mutagenic analysis of sFc further identifies key contact sites between the Fc-glycan and the Fc-backbone necessary to induce this conformational change and attain anti-inflammatory activity in vivo. These studies demonstrate that the conformational diversity of the Fc fragment serves as a general strategy to shift receptor specificity in order to effect different immunological outcomes.