Variable region heavy chain glycosylation determines the anticoagulant activity of a factor VIII antibody.
BACKGROUND
N-glycosylation occurs in the variable region of about 10% of antibodies but the role of carbohydrate at this location is still poorly understood.


OBJECTIVE
We investigated the function of N-glycosylation in the variable region of the heavy chain of a human monoclonal antibody, mAb-LE2E9, that partially inhibits factor VIII (FVIII) activity during coagulation.


RESULTS
Enzymatic deglycosylation indicated that the oligosaccharides do not determine the affinity of the antibody but enhance its FVIII neutralizing activity. A mutant antibody lacking the N-glycosylation site in the variable region of the heavy chain inhibited FVIII activity by up to 40%, while inhibition by the native antibody was 80%. To evaluate the physiological effect of such a FVIII inhibition, we investigated the ability of the mutant antibody devoid of N-glycosylation in the variable region to prevent thrombosis in mice with a strong prothombotic phenotype resulting from a type II deficiency mutation in the heparin binding site of antithrombin. Despite its moderate inhibition of FVIII activity, the mutant antibody significantly prevented thrombosis in treated animals. We also carried out glycan analysis of native and mutant antibodies.


CONCLUSIONS
Modification of glycosylation in the variable region of antibodies contributes to the diversity of FVIII type II inhibition possibly by steric hindrance of the active site of FVIII by glycans, and may provide a novel strategy to modulate the functional activity of therapeutic antibodies.
Jacquemin
M
M
Center for Molecular and Vascular Biology, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium. marc.jacquemin@med.kuleuven.ac.be
Radcliffe
C M
CM
Lavend'homme
R
R
Wormald
M R
MR
Vanderelst
L
L
Wallays
G
G
Dewaele
J
J
Collen
D
D
Vermylen
J
J
Dwek
R A
RA
Saint-Remy
J-M
JM
Rudd
P M
PM
Dewerchin
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D000925:Anticoagulants / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology*; D001483:Base Sequence; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D017931:DNA Primers; D005169:Factor VIII / Q000276:immunology*; D006031:Glycosylation; D006801:Humans; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D017931:DNA Primers; D005169:Factor VIII