Impact on N-Glycosylation profile of monoclonal anti-D antibodies as a way to control their immunoregulatory and cytotoxic properties

Abstract

Prophylaxis of hemolytic disease of newborns is based on the ability of polyclonal anti-D antibodies for suppressing maternal immune response against D-positive fetal red blood cells. The immunosuppressive effect of anti-D antibody is mediated by interaction between its Fc-fragment and low-affinity IgG Fc-receptor (FcγR) on the immune cell. No clinically effective monoclonal anti-D antibody (mAb) that can replace polyclonal anti-D immunoglobulin has been developed yet. The goals of this study were comparison of structural and functional properties of human anti-D polyclonal and monoclonal Abs and assessment of the possibility to manipulate the effector properties of the mAb. N-Glycosylation and particularly the content of nonfucosylated glycans are crucial for affinity of mAb to FcγRIIIA, which plays the key role in the clearance of sensitized cells. We studied and compared glycoprofiles and FcγRIIIA-mediated hemolytic ability of human polyclonal antibodies and anti-D mAbs produced by human B-cell lines, human-rodent heterohybridomas, and a human non-lymphoid cell line PER.C6. Replacement of producing cell line and use of glycosylation modulators can convert an inert mAb into an active one. Nevertheless, rodent cell lines, as well as human non-lymphoid cells, distort natural glycosylation of human IgG and could lead to the loss of immunosuppressive properties. All of the anti-D mAbs secreted by human B-cell lines have a glycoprofile close to human serum IgG. Hence, the constant ratio of IgG glycoforms in human serum is predetermined by glycosylation at the level of the individual antibody-producing cell. The anti-D fraction of polyclonal anti-D immunoglobulin compared to the total human IgG contains more nonfucosylated glycans. Thus, only human transformed B-cells are an appropriate source for efficient anti-D mAbs that can imitate the action of polyclonal anti-D IgG.

Antibody-Mediated Immune Suppression of Erythrocyte Alloimmunization Can Occur Independently from Red Cell Clearance or Epitope Masking in a Murine Model

“…Although not universally agreed upon, the most commonly accepted theories to explain the AMIS effect with erythrocytes are 1) that the AMIS Ab is able to rapidly clear Ag-positive target cells before they can be recognized by the immune system (10-12, 16, 17), theoretically based on FcgRmediated phagocytosis of D + RBCs preventing B cells from recognizing the D Ag (10,11,17), or 2) that the AMIS Ab sterically hinders the ability of the immune response to see or detect the Ag (4,15,16,18). Other theories with direct experimental support have suggested that inhibition may occur through the inhibitory FcgRIIB (19), that inhibitory cytokines may be produced (20), that glycosylation of the AMIS Ab may play a role (21)(22)(23), and/or that Abmediated immune deviation (AMID) away from the Ag and directed toward the sensitizing Ab may be involved (5). In addition to these theories, a number of others have been proposed (Table I).…”

“…In fact, regulators consider that for the prevention of HDFN a mAb specific for the RhD Ag should be demonstrated to cause RBC clearance before exposing pregnant women to the product. Along the lines of enhancing red cell clearance to derive an optimized mAb, additional attempts to biochemically increase the ability of some RhD-specific mAbs to cause increased red cell clearance characteristics by manipulating the carbohydrate structure on the Fc portion of the molecule have been performed (21,22). Unfortunately, we do not as of yet have a monoclonal anti-D Ab that has proved successful for the prevention of HDFN, and some anti-D Abs with good red cell clearance abilities have actually led to an enhanced alloimmune response rather than inhibition (22,23).…”

Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy

“…Afucosylated IgG has high affinity for FcγRIIIa 22,47 displacing plasma IgG and enabling ADCC at low concentrations 48 . Many alloantibodies, but not all, have considerably less fucose than total IgG1 [22][23][24][25]49 . Fucosylation of anti-D in 11 prophylactic preparations was 56%-91%, while for Rhophylac this was 81% 25 .…”

“…Methods used for other studies reported in Supplementary Table S1 were MALDI-TOF-MS analysis of IgG1 Fc-glycopeptides 25 , analysis by HVE-AEC, gel filtration chromatography and Concanavalin A binding of oligosaccharides released by hydrazinolysis 109 , quantitation of % G0 by binding of GlcNAc-specific mAb GN7 59 , chromatographic separation of fluorescently labelled neutral oligosaccharides 49 , enzymatically released glycans analysed by HPCE-LIF 82 and FAB-MS and MALDI-MS of permethylated N-glycans (Carbohydrate structure of rBRAD-3 and rBRAD-5; Joan Dalton, BioProducts Laboratory, UK, email, September 26, 2007; permission to publish subsequently given).…”

Quantitation of fetomaternal haemorrhage and F cells in unusual maternal blood samples by flow cytometry using anti‐D and anti‐HbF

“…Anti‐D immunoglobulin is currently derived from human plasma of immunised, boosted donors. Monoclonal anti‐D is not available as a substitute and recombinant anti‐D was ineffective in several trials (Kumpel, ), although development and glycosylation research is ongoing (Olovnikova et al , ; Yver et al , ).…”