Red blood cells (RBCs) from a rhesus D (RhD)-positive fetus that get in contact with immune cells of an RhD-negative pregnant woman during birth can induce a pathogenic antibody (Ab) response against the RhD-positive RBCs, leading to fetal hemolytic disease in subsequent pregnancies with RhD-positive fetuses after transplacental passage. To prevent allo-immunization by RhD-positive fetal RBCs, the RhD-negative mother receives one prenatal and one postnatal injection of serum immunoglobulin G (IgG) containing polyclonal RhD-specific IgG Abs that is purified from healthy RhD-negative men immunized with RhD-positive RBCs. Such a passive anti-RhD IgG Ab treatment i) induces a rapid clearance of RhD-positive RBCs from the bloodstream of the mother and ii) inhibits the development of pathogenic anti-RhD Abs by the mother ¹ .

However, the protective mechanism of passive anti-RhD treatment remains unclear. It is hypothesized that the clearance of RhD-positive RBCs is mediated through an Fcγ receptor (Fcγ R) IIIA-mediated Ab-dependent cellular cytotoxicity (ADCC) reaction and that rapid clearance prevents immunization ^{1– 4} . To prevent immunization, it has also been suggested that polyclonal anti-RhD IgG Abs have to inhibit the activation of RhD-specific B cells through the co-ligation of the B cell receptor and an inhibitory receptor, such as the IgG inhibitory receptor FcγRIIB ^{2, 3} . Furthermore, tolerance induction via antigen-presenting cells (APCs) has been posited to inhibit pro-inflammatory, RhD-specific T cell responses ^{2, 5, 6} . However, it is questionable whether FcγRIIIA crosslinking on APCs inhibits pro-inflammatory RhD-specific T cell responses but rather enforces pro-inflammatory T cell responses ⁷ . It is more likely that polyclonal anti-RhD IgG Abs target an inhibitory receptor (complex) on APCs to induce regulatory T cells and tolerance for inhibiting pro-inflammatory T cell and therewith also T cell-depemdent B cell responses.

Attempts to substitute this polyclonal anti-RhD IgG prophylaxis with RhD-specific monoclonal IgG Abs have failed because the monoclonal RhD-specific IgG Abs were relatively unstable due to intramolecular rearrangements or did not clear RhD-positive RBCs as rapidly as the available polyclonal anti-RhD IgG Abs in in vitro assays or clinical trials and/or did not sufficiently inhibit allo-immunization in clinical trials ^{1, 4, 8} .

It has become increasingly clear that the effector function of IgG Abs is highly regulated by the Abs’ Fc N-linked glycosylation pattern ( Figure 1A). Non-fucosylated (afucosylated) IgG Abs have a higher affinity for activating FcγRs, such as FcγRIIIA, and thus induce a stronger ADCC reaction than do fucosylated IgG Abs ^{9, 10} . This finding is currently translated, for example, into tumor-specific Ab therapy.

Agalactosylated and asialylated (G0), autoantigen-specific serum IgG Abs correlate with pro-inflammatory immune responses and disease activity in patients with rheumatoid arthritis (RA) ^{11– 14} . In contrast, pregnancy-induced and anti-tumor necrosis factor (TNF) therapy-induced remission in RA patients is associated with an increase in galactosylated and sialylated IgG Abs ^{15, 16} . In this context, an anti-inflammatory role has been suggested for sialylated IgG Abs. Accordingly, the sialylated IgG subfraction of intravenous IgG (IVIG), which is purified from pooled human plasma from healthy donors and used to systemically treat autoimmunity in high doses (2 g/kg), exhibits anti-inflammatory activity ^{17– 20} . We have recently shown that low doses of immune complexes (ICs) containing sialylated antigen-specific IgG Abs inhibit dendritic cell maturation and pro-inflammatory T and B cell immune responses in an antigen-specific manner ^{21– 23} . ICs containing 15%, but not 5%, sialylated IgG Abs have further been sufficient to inhibit B cell activation in vitro ²³ . Furthermore, it has recently been shown that ICs containing galactosylated, but not sialylated, IgG Abs are already sufficient to inhibit neutrophil activation ²⁴ .

Thus, G0 IgG Abs enhance, whereas galactosylated and sialylated IgG Abs suppress, pro-inflammatory immune responses. Accordingly, ICs containing galactosylated and sialylated IgG Abs inhibit rather than induce an ADCC reaction by at least reduced binding affinity of galactosylated and sialylated IgG Abs to FcγRIIIA ^{17, 25} but also likely by active suppression mechanisms via inhibitory receptors on immune cells.

However, based on the assumption that anti-RhD IgG Abs clear RhD-positive RBCs through an ADCC reaction, the different outcomes of monoclonal anti-RhD IgG Abs have been particularly attributed to different levels of fucose ^{1, 2, 4, 26– 29} . By contrast, the role of anti-RhD IgG galactosylation and sialylation has hardly been investigated.

Based on the idea that anti-tumor as well as anti-RhD IgG Abs should induce a strong ADCC response by recruiting FcγRIIIA-expressing immune cells, the French biotechnology company Laboratoire Francais du Fractionnement et des Biotechnologies (LFB; Les Ulis, France) has generated a monoclonal anti-CD20 IgG1 Ab (ublituximab; LFB-R603) and a human monoclonal RhD-specific IgG1 Ab (roledumab, LFB-R593 ³⁰ ) with low Fc fucosylation, low Fc galactosylation and low Fc sialylation based on their patent ³¹ . In the meantime the company has performed clinical phase I ³² and II (NCT00952575; completed 2011) studies on roledumab ³³ . The phase II study was designed to demonstrate the ability of roledumab to effectively eliminate exogenously administered RhD-positive RBCs from the circulation of an RhD-negative individual, thereby preventing RhD allo-immunization. The results have not been published yet.

However, based on the findings described above regarding the effector functions of differentially glycosylated IgG Abs, it is questionable whether an anti-tumor IgG1 Ab and an anti-RhD IgG1 Ab should have the same Fc glycosylation. Whether low-galactosylated, low-sialylated RhD-specific IgG Abs can inhibit the induction of pathogenic immune reactions against RhD-positive fetal RBCs or rather enhance allo-immunization is also questionable. To identify the Fc galactosylation and sialylation of RhD-specific IgG Abs in a commercially available polyclonal anti-RhD IgG product, we purified RhD-specific IgG Abs from the approved product Rhophylac® (CSL Behring, King of Prussia, PA, USA) and analyzed the Abs’ Fc glycosylation.

To assess the Fc galactosylation and sialylation of RhD-specific IgG Abs in a commercially available polyclonal anti-RhD IgG product, we purified RhD-specific IgG Abs from Rhophylac® and analyzed the Abs’ Fc glycosylation ( Figure 1, Supplementary figure 1 and Supplementary figure 2 and the data files). We found that the purified polyclonal RhD-specific IgG Abs were even more galactosylated and sialylated than the total Rhophylac® IgG Abs ( Figure 1, Supplementary figure 1 and Supplementary figure 2 and the data files), which are comparable to total IgG Abs in immunosuppressive IVIG. The injection of RhD-positive RBCs into RhD-negative men obviously induced no pathogenic immune response. Comparable results have recently been generated by analyzing allergen-specific human IgG Abs after successful allergen-specific immunotherapy for birch pollen allergy ²¹ . Together, these results further indicate that tolerance induction induces mainly galactosylated and sialylated IgG Abs that have the potential to inhibit pathogenic immune responses via ICs ^{21– 24} .

These results strongly suggest that only galactosylated and sialylated, immunosuppressive monoclonal RhD-specific IgG Abs can be substituted for polyclonal anti-RhD IgG products to inhibit pathogenic allo-immunity in RhD-negative pregnant woman. Low fucosylated, low agalactosylated and low sialylated RhD-specific IgG Abs might not only enhance pro-inflammatory immune responses against RhD-positive fetal RBCs but also have the potential to attack RhD-positive fetal RBCs after transplacental passage.