Enzymatic deglycosylation converts pathogenic neuromyelitis optica anti-aquaporin-4 immunoglobulin G into therapeutic antibody - PubMed (original) (raw)

Enzymatic deglycosylation converts pathogenic neuromyelitis optica anti-aquaporin-4 immunoglobulin G into therapeutic antibody

Lukmanee Tradtrantip et al. Ann Neurol. 2013 Jan.

Abstract

Objective: Neuromyelitis optica (NMO) is caused by binding of pathogenic autoantibodies (NMO-immunoglobulin G [IgG]) to aquaporin-4 (AQP4) on astrocytes, which initiates complement-dependent cytotoxicity (CDC) and inflammation. We recently introduced mutated antibody (aquaporumab) and small-molecule blocker strategies for therapy of NMO, based on prevention of NMO-IgG binding to AQP4. Here, we investigated an alternative strategy involving neutralization of NMO-IgG effector function by selective IgG heavy-chain deglycosylation with bacteria-derived endoglycosidase S (EndoS).

Methods: Cytotoxicity and NMO pathology were measured in cell and spinal cord slice cultures, and in mice exposed to control or EndoS-treated NMO-IgG.

Results: EndoS treatment of NMO patient serum reduced by >95% CDC and antibody-dependent cell-mediated cytotoxicity, without impairment of NMO-IgG binding to AQP4. Cytotoxicity was also prevented by addition of EndoS after NMO-IgG binding to AQP4. The EndoS-treated, nonpathogenic NMO-IgG competitively displaced pathogenic NMO-IgG bound to AQP4, and prevented NMO pathology in spinal cord slice culture and mouse models of NMO.

Interpretation: EndoS deglycosylation converts pathogenic NMO-IgG autoantibodies into therapeutic blocking antibodies. EndoS treatment of blood may be beneficial in NMO, and may be accomplished, for example, by therapeutic apheresis using surface-immobilized EndoS.

Copyright © 2012 American Neurological Association.

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Conflict of interest statement

Potential Conflicts of Interest. Drs. Verkman and Tradtrantip are named co-inventors on a patent application filed on EndoS therapy of NMO. The intellectual property is owned by the University of California.

Figures

Figure 1

Figure 1. EndoS deglycosylation of NMO-IgG prevents CDC and ADCC

A. (left) Schematic of IgG showing the Fc glycosylation at Asn-297, and Fab binding to AQP4. (right) Sugar moiety at Asn-297 with EndoS cleavage site shown. Asn, asparagine; Fuc, fucose; GlcNac, N-acetylglucosamine; Man, mannose; Gal, galactose; Sial, sialic acid. B. Commassie blue SDS-PAGE and Lens culinaris agglutinin (LCA) lectin blot of control and EndoS-treated NMO-IgG and purified IgG from NMO sera. C. (top) CDC in AQP4-expressing CHO and U87MG cells incubated with NMO-IgG or NMO-IgGGL− and 2% human complement, as quantified by LDH release (S.E., n=4). (bottom) Live/dead (green/red) staining of AQP4-expressing CHO cells incubated with 5 μg/mL NMO-IgG or NMO-IgGGL− and 2% human complement. D. ADCC in AQP4-expressing CHO cells incubated with NK-cells and 20 μg/mL NMO-IgG or NMO-IgGGL−, as quantified by percentage dead cells (S.E., n=4). (bottom) Live/dead (green/red) staining.

Figure 2

Figure 2. EndoS deglycosylation of NMO serum prevents CDC and ADCC

A. (left) CDC in AQP4-expressing CHO cells incubated with NMO serum or NMO serumGL− and 2% human complement, as quantified by LDH release (top) and live/dead staining (bottom). (right) Summary of data from three NMO sera (S.E., n=6, P < 0.001). B. ADCC in AQP4-expressing CHO cells incubated with NK-cells and control or EndoS-treated IgG from NMO sera (1 mg/mL), as quantified by percentage dead cells (S.E., n=5, P < 0.001). (bottom) Live/dead (green/red) staining. C. EndoS addition in situ after NMO-IgG binding to AQP4 reduces CDC. CDC was measured by LDH release in AQP4-expressing CHO cells incubated with NMO serum for 30 min, then treated with EndoS for 30 min, followed by 2% human complement for 1 h. (S.E., n=4, * P < 0.01).

Figure 3

Figure 3. EndoS-treated NMO-IgG binds to AQP4 and competes with binding of pathogenic NMO-IgG

A. Binding of NMO-IgG to AQP4 in CHO cells. (left) Fluorescence micrographs of AQP4-expressing CHO cells stained for NMO-IgG or NMO-IgGGL− (red) and AQP4 (green). (right) Binding of NMO-IgG and NMO-IgGGL− showing red-to-green fluorescence ratio (R/G) as a function of NMO-IgG concentration (S.E., n=3). Differences not significant. B. Binding of control and EndoS-treated NMO patient serum to AQP4 on CHO cells. C. EndoS-treated NMO-IgG protects against CDC caused by (untreated) NMO-IgG. LDH release assayed in CHO cells after 1 h incubation with indicated concentrations of NMO-IgG and NMO-IgGGL−, together with 2% human complement.

Figure 4

Figure 4. EndoS treatment prevents lesions in an ex vivo spinal cord slice culture model of NMO

A. Spinal cord slice cultures were exposed to 5 μg/mL NMO-IgG or NMO-IgGGL− and 5% human complement (HC). Representative GFAP and AQP4 immunofluorescence shown after 24 h. B. Summary of lesion scores from experiments as in A (S.E., 6 slices per group, * P < 0.01). C. Slice cultures were incubated with 5 μg/mL NMO-IgG, and then 30 min later with 20 U/mL EndoS, and 5 % HC added 60 min later. D. Lesion scores (S.E., 6 slices per group, * P < 0.01).

Figure 5

Figure 5. EndoS treatment prevents lesions in an in vivo mouse model of NMO

A. Brains of live mice were injected with 0.6 μg NMO-IgG or NMO-IgGGL− together with 3 μL human complement (HC). Representative GFAP, AQP4 and myelin (MBP) immunofluorescence at 3 days after injection. Yellow line represents needle tract. White line delimits the lesion with loss of AQP4, GFAP and myelin. B. Higher magnification of brains injected with NMO-IgG and HC. White dashed line delimits the lesion (top). Contralateral hemispheres (non-injected) are shown (right). C. Summary of lesion size from experiments as in A (S.E., 4 mice per group, * P < 0.01 by the non parametric Mann-Whitney test). D. Brains were injected with 30 μg of purified IgG from NMO serum and 105 μg of EndoS-treated IgG purified from the same NMO patient (NMO serumGL−) or a non-NMO control (control serumGL−), together with 3 μL HC. (left) Representative GFAP, AQP4 and MBP immunofluorescence at 3 days after injection. Yellow line shows the needle tract and white line delimits the lesion. (right) Summary lesion size (S.E., 4 mice per group, * P < 0.01 by the non parametric Mann-Whitney test).

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