Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy (original) (raw)
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Chapter Low but highly variable Fc-fucosylation of anti-D IgG 1 in pregnancy
2014
Hemolytic disease of the fetus and newborn (HFDN) may occur when maternal anti-D IgG antibodies cross the placenta and mediate the destruction of D-positive red blood cells (RBCs) via phagocytic-IgG Fc-receptors (FcγR). Clinical severity is not strictly related to titer, but is more accurately predicted by functional antibody-dependent cellular cytotoxicity (ADCC), suggesting other factors to play a role. Since the composition of the N-linked glycan at position 297 in the Fc-region of IgG1 is critical for binding to FcγR, we analyzed IgG-derived glyco-peptides from anti-D IgG1 purified from plasma of alloimmunized pregnant women by mass spectrometry. Seventy pregnancy-induced anti-D samples, taken from different women, revealed a varied, but strong decrease in Fc-fucosylation in the majority of anti-D IgG1, but not in total IgG. The degree of anti-D fucosylation correlated significantly with CD16 (FcγRIIIa)-mediated antibody-dependent cellular cytotoxicity (ADCC) by NK-cells, in agr...
Low but highly variable Fc-fucosylation of anti-D IgG1 in pregnancy
2014
Hemolytic disease of the fetus and newborn (HFDN) may occur when maternal anti-D IgG antibodies cross the placenta and mediate the destruction of D-positive red blood cells (RBCs) via phagocytic-IgG Fc-receptors (FcγR). Clinical severity is not strictly related to titer, but is more accurately predicted by functional antibody-dependent cellular cytotoxicity (ADCC), suggesting other factors to play a role. Since the composition of the N-linked glycan at position 297 in the Fc-region of IgG1 is critical for binding to FcγR, we analyzed IgG-derived glyco-peptides from anti-D IgG1 purified from plasma of alloimmunized pregnant women by mass spectrometry. Seventy pregnancy-induced anti-D samples, taken from different women, revealed a varied, but strong decrease in Fc-fucosylation in the majority of anti-D IgG1, but not in total IgG. The degree of anti-D fucosylation correlated significantly with CD16 (FcγRIIIa)-mediated antibody-dependent cellular cytotoxicity (ADCC) by NK-cells, in agr...
Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D
Transfusion, 2014
BACKGROUND: RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with FcγRIII, with less Fc-fucosylation strengthening the interaction.
British Journal of Haematology
Haemolytic disease of the fetus and newborn (HDFN) is a severe disease in which fetal red blood cells (RBC) are destroyed by maternal anti-RBC IgG alloantibodies. HDFN is most often caused by anti-D but may also occur due to anti-K,-c-orE. We recently found N-linked glycosylation of anti-D to be skewed towards low fucosylation, thereby increasing the affinity to IgG-Fc receptor IIIa and IIIb, which correlated with HDFN disease severity. Here, we analysed 230 pregnant women with anti-c,-E or-K alloantibodies from a prospective screening cohort and investigated the type of Fc-tail glycosylation of these antibodies in relation to the trigger of immunisation and pregnancy outcome. Anti-c,-E and-K showindependent of the event that had led to immunisationa different kind of Fc-glycosylation compared to that of the total IgG fraction, but with less pronounced differences compared to anti-D. High Fc-galactosylation and sialylation of anti-c correlated with HDFN disease severity, while low anti-K Fc-fucosylation correlated with severe fetal anaemia. IgG-Fc glycosylation of anti-RBC antibodies is shaped depending on the antigen. These features influence their clinical potency and may therefore be used to predict severity and identify those needing treatment.
Comparison of the Fc glycosylation of fetal and maternal immunoglobulin G
Glycoconjugate Journal, 2013
Human immunoglobulin G (IgG) molecules are composed of two Fab portions and one Fc portion. The glycans attached to the Fc portions of IgG are known to modulate its biological activity as they influence interaction with both complement and various cellular Fc receptors. IgG glycosylation changes significantly with pregnancy, showing a vast increase in galactosylation and sialylation and a concomitant decrease in the incidence of bisecting GlcNAc. Maternal IgGs are actively transported to the fetus by the neonatal Fc receptor (FcRn) expressed in syncytiotrophoblasts in the placenta, providing the fetus and newborn with immunological protection. Two earlier reports described significant differences in total glycosylation between fetal and maternal IgG, suggesting a possible glycosylationselective transport via the placenta. These results might suggest an alternative maternal transport pathway, since FcRn binding to IgG does not depend on Fc-glycosylation. These early studies were performed by releasing N-glycans from total IgG. Here, we chose for an alternative approach analyzing IgG Fc glycosylation at the glycopeptide level in an Fcspecific manner, providing glycosylation profiles for IgG1 and IgG4 as well as combined Fc glycosylation profiles of IgG2 and 3. The analysis of ten pairs of fetal and maternal IgG samples revealed largely comparable Fc glycosylation for all the analyzed subclasses. Average levels of galactosylation, sialylation, bisecting GlcNAc and fucosylation were very similar for the fetal and maternal IgGs. Our data suggest that the placental IgG transport is not Fc glycosylation selective.
Isolation and characterization of IgG1 with asymmetrical Fc glycosylation
Glycobiology, 2011
N-glycosylation of immunoglobulin G (IgG) at asparigine residue 297 plays a critical role in antibody stability and immune cell-mediated Fc effector function. Current understanding pertaining to Fc glycosylation is based on studies with IgGs that are either fully glycosylated [both heavy chain (HC) glycosylated] or aglycosylated (neither HC glycosylated). No study has been reported on the properties of hemi-glycosylated IgGs, antibodies with asymmetrical glycosylation in the Fc region such that one HC is glycosylated and the other is aglycosylated. We report here for the first time a detailed study of how hemiglycosylation affects the stability and functional activities of an IgG1 antibody, mAb-X, in comparison to its fully glycosylated counterpart. Our results show that hemi-glycosylation does not impact Fab-mediated antigen binding, nor does it impact neonatal Fc receptor binding. Hemi-glycosylated mAb-X has slightly decreased thermal stability in the CH2 domain and a moderate decrease ( 20%) in C1q binding. More importantly, the hemi-glycosylated form shows significantly decreased binding affinities toward all Fc gamma receptors (FcγRs) including the high-affinity FcγRI, and the low-affinity FcγRIIA, FcγRIIB, FcγRIIIA and FcγRIIIB. The decreased binding affinities to FcγRs result in a 3.5-fold decrease in antibody-dependent cell cytotoxicity (ADCC). As ADCC often plays an important role in therapeutic antibody efficacy, glycosylation status will not only affect the antibody quality but also may impact the biological function of the product.
The glycosylation pattern of humanized IgGI antibody (D1.3) expressed in CHO cells
Glycoconjugate journal, 1997
A humanized IgG antibody (D1.3) which retains murine complementarity determining regions specific for the antigen lysozyme has been expressed in CHO-DUKX cells. Heavy and light chain containing plasmids were co-transfected into CHO-DUKX cells and stable clones were grown in DMEM/F12 medium supplemented with 5% foetal calf serum. D1.3 antibody was purified from culture supernatants by Protein G chromatography. With the recombinant D1.3 antibody as a model, this cell culture system was shown to glycosylate the IgG Fc region in a similar manner to IgG isolated from serum. The neutral, core fucosylated biantennary oligosaccharides found are present in serum IgG and no novel carbohydrate sequences were detected. The degree of terminal agalactosylation was also similar to normal serum, in contrast to the increased levels found in rheumatoid serum. Furthermore, those oligosaccharides which lack only one terminal Gal are exclusively galactosylated on the GlcNAc(beta1,2) Man(alpha1,6) Man(be...
Functional in vitro studies of recombinant human immunoglobulin�G and immunoglobulin�A anti-D
Transfusion, 2007
The use of anti-D purified from human serum to prevent hemolytic disease of the fetus and newborn due to D is well established. Owing to supply and safety reasons, however, an unlimited and nonplasma-derived source of antibodies for Rhesus prophylaxis is needed. STUDY DESIGN AND METHODS: Recombinant human immunoglobulin G (IgG)1, IgG2, IgG3, IgG4, IgA1, and IgA2 anti-D with the same variable region were expressed in Chinese hamster ovary cells. The effector functions of these antibodies were assessed by an antibody-dependent cell-mediated cytotoxicity (ADCC) assay and a chemiluminescence (CL) method for detection of respiratory burst. RESULTS: In the ADCC assay, IgG1, IgG3, and IgA1 did the best and were as active as a currently used prophylactic polyclonal anti-D. IgG4 and IgA2 were moderately active, whereas IgG2 was not active. In the CL assay, IgG1 and IgG3 were active but much less so than a currently used prophylactic polyclonal anti-D. For some effector cell preparations, IgG4 was active in the CL assay, whereas IgG2, IgA1, and IgA2 were not. A mixture of IgG1 and IgG3 showed a synergistic effect in the CL assay and did as well as the prophylactic polyclonal anti-D in ADCC and CL. Mixtures of IgA1 and either IgG1 or IgG3 showed no synergistic effect. CONCLUSION: A mixture of recombinant human IgG1 and IgG3 anti-D could be of value in future Rhesus prophylaxis.
Glycosylation and functional activity of anti-D secreted by two human lymphoblastoid cell lines
Cytotechnology, 1994
Cell lines BTSN4 and BTSN5 were produced by the Epstein-Barr Virus (EBV) transformation of B-lymphocytes from the same human donor. Both secrete an anti-D monoclonal of the IgG1 subclass but these antibodies display vastly different effector activities. Specifically, anti-D from BTSN4 has a far greater activity in both monocyteand lymphocyte-mediated ADCC reactions and causes a higher percentage of rosettes to be formed with monocyte-like U937 cells. This variation in functional activity is shown to coincide with changes in the structure of the sugar chains attached to the asparagine-297 site on the immunoglobulin heavy chain.