Reduced FcRn-mediated transcytosis of IgG2 due to a missing Glycine in its lower hinge.

Neonatal Fc-receptor (FcRn), the major histocompatibility complex (MHC) class I-like Fc-receptor, transports immunoglobuline G (IgG) across cell layers, extending IgG half-life in circulation and providing newborns with humoral immunity. IgG1 and IgG2 have similar half-lives, yet IgG2 displays lower foetal than maternal concentration at term, despite all known FcRn binding residues being preserved between IgG1 and IgG2. We investigated FcRn mediated transcytosis of VH-matched IgG1 and IgG2 and mutated variants thereof lacking Fc-gamma receptor (FcγR) binding in human cells expressing FcRn. We observed that FcγR binding was not required for transport and that FcRn transported less IgG2 than IgG1. Transport of IgG1 with a shortened lower hinge (ΔGly236, absent in germline IgG2), was reduced to levels equivalent to IgG2. Conversely, transport of IgG2 + Gly236 was increased to IgG1 levels. Gly236 is not a contact residue between IgG and FcRn, suggesting that its absence leads to an altered conformation of IgG, possibly due to a less flexible Fab, positioned closer to the Fc portion. This may sterically hinder FcRn binding and transport. We conclude that the lack of Gly236 is sufficient to explain the reduced FcRn-mediated IgG2 transcytosis and accounts for the low maternal/fetal IgG2 ratio at term.

Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport.

We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders. Here, we show that G1Δnab mutants retain pH-dependent binding to human FcRn but that the amino acid alterations reduce the affinity of the IgG1:FcRn interaction by 2.0-fold and 1.6-fold for the two antibodies investigated. The transport of the modified G1Δnab mutants across monolayers of human cell lines expressing FcRn was approximately 75% of the wild-type, except that no difference was observed with human umbilical vein endothelial cells. G1Δnab mutation also reduced transport in an ex vivo placenta model. In conclusion, we demonstrate that, although the G1Δnab mutations are away from the FcRn-binding site, they have long-distance effects, modulating FcRn binding and transcellular transport. Our findings have implications for the design of therapeutic human IgG with tailored effector functions.

The multiple facets of FcRn in immunity.

The neonatal Fc receptor, FcRn, is best known for its role in transporting IgG in various tissues, providing newborns with humoral immunity, and for prolonging the half-life of IgG. Recent findings implicate the involvement of FcRn in a far wider range of biological and immunological processes, as FcRn has been found to bind and extend the half-life of albumin; to be involved in IgG transport and antigen sampling at mucosal surfaces; and to be crucial for efficient IgG-mediated phagocytosis. Herein, the function of FcRn will be reviewed, with emphasis on its recently documented significance for IgG polymorphisms affecting the half-life and biodistribution of IgG3, on its role in phagocyte biology, and the subsequent role for the presentation of antigens to lymphocytes.

On the perplexingly low rate of transport of IgG2 across the human placenta.

The neonatal receptor, FcRn, mediates both serum half-life extension as well as active transport of maternal IgG to the fetus during pregnancy. Therefore, transport efficiency and half-life go hand-in-hand. However, while the half-life of the human IgG2 subclass is comparable to IgG1, the placental transport of IgG2 is not, with the neonatal IgG1 levels generally exceeding maternal levels at birth, but not for IgG2. We hypothesized that the unique short-hinged structure of IgG2, which enables its κ-, but not λ-isotype to form at least three different structural isoforms, might be a contributing factor to these differences. To investigate whether there was any preference for either light chain, we measured placental transport of IgG subclasses as well as κ/λ-light chain isotypes of IgG1 and IgG2 in 27 matched mother-child pairs. We also studied the half-life of IgG1 and IgG2 light chain isotypes in mice, as well as that of synthesized IgG2 structural isotypes κA and κB. In order to investigate serum clearance of IgG1 and IgG2 light-chain isotypes in humans, we quantified the relative proportions of IgG1 and IgG2 light chains in hypogammaglobulinemia patients four weeks after IVIg infusion and compared to the original IVIg isotype composition. None of our results indicate any light chain preference in either of the FcRn mediated mechanisms; half-life extension or maternal transport.

Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential.

Human IgG3 displays the strongest effector functions of all IgG subclasses but has a short half-life for unresolved reasons. Here we show that IgG3 binds to IgG-salvage receptor (FcRn), but that FcRn-mediated transport and rescue of IgG3 is inhibited in the presence of IgG1 due to intracellular competition between IgG1 and IgG3. We reveal that this occurs because of a single amino acid difference at position 435, where IgG3 has an arginine instead of the histidine found in all other IgG subclasses. While the presence of R435 in IgG increases binding to FcRn at neutral pH, it decreases binding at acidic pH, affecting the rescue efficiency-but only in the presence of H435-IgG. Importantly, we show that in humans the half-life of the H435-containing IgG3 allotype is comparable to IgG1. H435-IgG3 also gave enhanced protection against a pneumococcal challenge in mice, demonstrating H435-IgG3 to be a candidate for monoclonal antibody therapies.

FcRn: an IgG receptor on phagocytes with a novel role in phagocytosis.

Here, we report that the MHC class I-related neonatal Fc receptor (FcRn) is expressed within azurophilic and specific granules of neutrophils and relocates to phagolysosomes on phagocytosis of IgG-opsonized bacteria. We found FcRn to enhance phagocytosis in a pH-dependent manner which was independent of IgG recycling. IgG-opsonized bacteria were inefficiently phagocytosed by neutrophils from beta2M knock-out or FcRn alpha-chain knock-out mice, which both lack expression of FcRn. Similarly, low phagocytic activity was also observed with mutated IgG (H435A), which is incapable of binding to FcRn, while retaining normal binding to classical leukocyte Fcgamma receptors. Finally, a TAT peptide representing intracellular endocytosis and transport motifs within FcRn strongly inhibited IgG-mediated phagocytosis. These findings support a novel concept in which FcRn fulfills a major role in IgG-mediated phagocytosis.