Effector Properties and Glycosylation Patterns of Recombinant Human Anti-D-IgG1 Antibodies Produced by Human PER.C6® Cells

Abstract

Creation of effective monoclonal anti-D immunoglobulin for prevention of hemolytic disease of the newborn remains an unsolved problem because there is still no producer cell strain providing stable production and adequate glycosylation of antibodies. Recombinant anti-D have been obtained on the basis of human PER.C6(®) cells and characterized. Anti-D antibodies expressed in PER.C6(®) exhibited lower hemolytic activity in antibody-dependent cytotoxicity (ADCC) reaction mediated by low-affinity Fcγ receptors in comparison with identical antibodies of lymphoblastoid origin. Monoclonal antibodies produced by PER.C6(®) are completely fucosylated and desialylated, i.e. are characterized by abnormal glycosylation. Addition of kifunensine (α-mannosidase I inhibitor) to the medium led to production of antibodies with high hemolytic activity. Reduced activity of monoclonal antibodies in PER.C6(®) cells and the effect of kifunensine (causing synthesis of defucosylated glycans) suggest that the absence of fucose is the key factor responsible for Fc affinity for low-affinity receptors.

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).…”

Glycosylation in Cell Culture

“…It has been used to produce IgG with high amounts of fucosylated mono-galactosylated (FG1) glycan, similar to serum IgG (Jones et al 2003). However, another monoclonal antibody produced in PER.C6® had much lower galactosylation with 60 % of the Mab agalactosylated (FG0) (Olovnikova et al 2012), suggesting it may not produce similar glycosylation for all recombinant products, although differences in culture conditions could account for some variability. Human alpha-1-antitrypsin produced in PER.C6® had increased core fucosylation, increased tri-and tetraantennary glycans and changes in microheterogeneity at specific sequons compared to the plasma derived protein .…”