Abstract
Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo.
Highlights
Immunoglobulin G (IgG) antibodies are glycoproteins with pro- and anti-inflammatory effector functions
The genetic heterogeneity of the human population and age dependent alterations of immune responses may further complicate to predict how stable therapeutic antibodies are in individual patients and with respect to glycosylation. To address this issue and identify to what extent therapeutic IgG preparations are subject to B cell independent sialylation, we made use of two mouse strains lacking either B cells or ST6Gal1, which is the responsible enzyme for adding terminal sialic acid residues to the IgG sugar moiety [23,24,25]
An important step in the process of introducing a new therapeutic IgG antibody or a new batch of an already approved antibody into the clinic is an in-depth characterization of the IgG glycovariants present in the antibody preparation. It is well-known that many factors, such as the cell line in which a therapeutic antibody is produced [43] or the specific culture conditions [44], can alter IgG glycosylation
Summary
Immunoglobulin G (IgG) antibodies are glycoproteins with pro- and anti-inflammatory effector functions. While desialylated IVIg lost its capacity to suppress a wide variety of autoimmune diseases in mice [12, 13], IVIg preparations enriched for terminal sialic acid residues showed an enhanced anti-inflammatory activity [5, 14]. Of note, enriching cytotoxic antibodies for terminal sialic acid residues decreased their activity in vivo and in vitro in some but not all studies [15]. Consistent with this reduced activity a reduction in affinity of highly sialylated IgGs for select activating FcγRs was noted [5]. Due to the potent immune modulating functions of select IgG glycoforms, new therapeutic approaches try to alter IgG activity by modulating its glycosylation ex vivo [5, 16], or by changing the glycosylation status in the patient by enzymatic approaches [17,18,19]
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