3 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 Ã…, 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
Chemical and Structural Analysis of an Antibody Folding Intermediate Trapped during Glycan Biosynthesis
Human IgG Fc glycosylation modulates immunological effector
functions
such as antibody-dependent cellular cytotoxicity and phagocytosis.
Engineering of Fc glycans therefore enables fine-tuning of the therapeutic
properties of monoclonal antibodies. The N-linked glycans of Fc are
typically complex-type, forming a network of noncovalent interactions
along the protein surface of the Cγ2 domain. Here, we manipulate
the mammalian glycan-processing pathway to trap IgG1 Fc at sequential
stages of maturation, from oligomannose- to hybrid- to complex-type
glycans, and show that the Fc is structurally stabilized following
the transition of glycans from their hybrid- to complex-type state.
X-ray crystallographic analysis of this hybrid-type intermediate reveals
that N-linked glycans undergo conformational changes upon maturation,
including a flip within the trimannosyl core. Our crystal structure
of this intermediate reveals a molecular basis for antibody biogenesis
and provides a template for the structure-guided engineering of the
protein–glycan interface of therapeutic antibodies
Chemical and Structural Analysis of an Antibody Folding Intermediate Trapped during Glycan Biosynthesis
Human IgG Fc glycosylation modulates immunological effector
functions
such as antibody-dependent cellular cytotoxicity and phagocytosis.
Engineering of Fc glycans therefore enables fine-tuning of the therapeutic
properties of monoclonal antibodies. The N-linked glycans of Fc are
typically complex-type, forming a network of noncovalent interactions
along the protein surface of the Cγ2 domain. Here, we manipulate
the mammalian glycan-processing pathway to trap IgG1 Fc at sequential
stages of maturation, from oligomannose- to hybrid- to complex-type
glycans, and show that the Fc is structurally stabilized following
the transition of glycans from their hybrid- to complex-type state.
X-ray crystallographic analysis of this hybrid-type intermediate reveals
that N-linked glycans undergo conformational changes upon maturation,
including a flip within the trimannosyl core. Our crystal structure
of this intermediate reveals a molecular basis for antibody biogenesis
and provides a template for the structure-guided engineering of the
protein–glycan interface of therapeutic antibodies