18 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
Efficacy of <i>N</i>B-DNJ in guinea pigs challenged with EBOV.
<p>Female guinea pigs were infected with 10<sup>3</sup> pfu of EBOV (Zaire strain) via IV cannula. Animals were untreated (n = 3), treated IV TID (8 hourly) with 1850 mg/kg/day <i>N</i>B-DNJ (n = 6) or placebo (n = 3) for 14 days. (A) Percentage of surviving animals. (B) Signs of illness. (C) Body temperature, compared to their temperature on day 0 (baseline) and (D) weight change as a percentage of body weight on day 0 (baseline). Mean for each group +/- standard deviation is plotted. Note: Temperatures in panel C show means of only 5 out of the 6 animals treated with <i>N</i>B-DNJ due to the temperature chip failing in one of the animals in this group.</p
Severity of histological lesions in samples of EBOV challenged animals after treatment (second efficacy study).
<p>Severity of histological lesions in samples of EBOV challenged animals after treatment (second efficacy study).</p
Effect of iminosugar treatment on guinea pig weight.
<p>Female guinea pigs were treated via IV cannula TID with 1850 mg/kg/day <i>N</i>B-DNJ (n = 6), 120 mg/kg/day M<i>O</i>N-DNJ (n = 4) or placebo (n = 4) for 16 days. Weight change as a percentage of body weight on day 0 (baseline) is shown for placebo (black circle), <i>N</i>B-DNJ (blue square, including euthanised animal) and M<i>O</i>N-DNJ (orange triangle) treatment groups. Means for each group +/- standard deviation are plotted.</p
Efficacy of <i>N</i>B-DNJ and M<i>O</i>N-DNJ in guinea pigs challenged with EBOV.
<p>Female guinea pigs were infected with 10<sup>3</sup> pfu of EBOV (Zaire strain) via IV cannula then treated by the same route TID (between 9am and 5pm) with 1850 mg/kg/day <i>N</i>B-DNJ (n = 4), 120 mg/kg/day M<i>O</i>N-DNJ (n = 4) or placebo (n = 4) for 14 days. (A) Percentage of surviving animals. (B) Signs of illness. (C) Body temperature, compared to their temperature on day 0 (baseline) and (D) weight change as a percentage of body weight on day 0 (baseline). Mean for remaining animals in each group plotted for B, C and D. Asterisks indicate significantly different weights (p < 0.05; Mann-Whitney test) between animals in placebo and <i>N</i>B-DNJ-treated groups at day 3 (p = 0.0304) and in <i>N</i>B-DNJ- and M<i>O</i>N-DNJ-treated groups at day 4 (p = 0.0304) and day 5 (p = 0.0304).</p
Production of EBOV glycoprotein pseudotyped virus in the presence or absence of iminosugars does not alter infectivity.
<p>EBOV glycoprotein pseudotyped lentivirus was produced in the presence of 100μM of <i>N</i>B-DNJ, <i>N</i>N-DNJ, <i>N</i>B-DGJ or <i>N</i>N-DGJ, equivalent DMSO or media alone. The infectivity of these viruses was then assayed in HEK 293T cells in the (A) presence or (B) absence of homologous drug and recorded as luciferase reporter gene expression. The NIBSC international serological standard was included as a positive control for neutralisation. Pseudotyped virus production was undertaken twice, each time infections were performed in duplicate, and data analysed together. Average RLU are shown. For clarity the SD error is shown for only media and DMSO data but these are representative of all data sets. Single one phase exponential curves were fitted to the <i>N</i>B-DNJ, <i>N</i>B-DGJ and media data (solid line) and to <i>N</i>N-DNJ, <i>N</i>N-DGJ and DMSO data (dashed line).</p
Severity of histological lesions in HE stained tissues from iminosugar treated guinea pigs, challenged with EBOV.
<p>Severity of histological lesions in HE stained tissues from iminosugar treated guinea pigs, challenged with EBOV.</p
Histopathological observations in Ebola virus-challenge guinea pigs.
<p>(A) Liver, placebo animal 1. Hepatocyte vacuolation and a focus of necrosis (arrow). (B) Liver, placebo animal 1. Hepatocyte necrosis and mineralisation of necrotic cells. (C) Liver, M<i>O</i>N-DNJ-treated animal 11. Necrosis and depletion of hepatocytes from around a portal triad. (D) Spleen, M<i>O</i>N-DNJ-treated animal 10. Scattered necrotic/apoptotic cells and macrophage-like cells in the red pulp at the interface with the marginal zone.</p
Summary of <i>in vitro</i> antiviral properties of iminosugars.
<p>Summary of <i>in vitro</i> antiviral properties of iminosugars.</p
<i>In vitro</i> enzyme inhibition of iminosugars.
<p><i>In vitro</i> enzyme inhibition of iminosugars.</p