1 research outputs found
Engineering Hydrophobic Protein–Carbohydrate Interactions to Fine-Tune Monoclonal Antibodies
Biologically active
conformations of the IgG1 Fc homodimer are
maintained by multiple hydrophobic interactions between the protein
surface and the N-glycan. The Fc glycan modulates biological effector
functions, including antibody-dependent cellular cytotoxicity (ADCC)
which is mediated in part through the activatory Fc receptor, FcγRIIIA.
Consistent with previous reports, we found that site-directed mutations
disrupting the protein–carbohydrate interface (F241A, F243A,
V262E, and V264E) increased galactosylation and sialylation of the
Fc and, concomitantly, reduced the affinity for FcγRIIIA. We
rationalized this effect by crystallographic analysis of the IgG1
Fc F241A mutant, determined here to a resolution of 1.9 Å, which
revealed localized destabilization of this glycan–protein interface.
Given that sialylation of Fc glycans decreases ADCC, one explanation
for the effect of these mutants on FcγRIIIA binding is their
increased sialylation. However, a glycan-engineered IgG1 with hypergalactosylated
and hypersialylated glycans exhibited unchanged binding affinity to
FcγRIIIA. Moreover, when we expressed these mutants as a chemically
uniform (Man<sub>5</sub>GlcNAc<sub>2</sub>) glycoform, the individual
effect of each mutation on FcγRIIIA affinity was preserved.
This effect was broadly recapitulated for other Fc receptors (FcγRI,
FcγRIIA, FcγRIIB, and FcγRIIIB). These data indicate
that destabilization of the glycan–protein interactions, rather
than increased galactosylation and sialylation, modifies the Fc conformation(s)
relevant for FcγR binding. Engineering of the protein–carbohydrate
interface thus provides an independent parameter in the engineering
of Fc effector functions and a route to the synthesis of new classes
of Fc domain with novel combinations of affinities for activatory
and inhibitory Fc receptors