Electron tomography of late stages of FcRn-mediated antibody transcytosis in neonatal rat small intestine

The neonatal Fc receptor (FcRn) transports maternal immunoglobulin (IgG) across epithelia to confer passive immunity to mammalian young. In newborn rodents, FcRn transcytoses IgG from ingested milk across the intestinal epithelium for release into the bloodstream. We used electron tomography to examine FcRn transport of Nanogold-labeled Fc (Au-Fc) in neonatal rat jejunum, focusing on later aspects of transport by chasing Au-Fc before fixation. We observed pools of Au-Fc in dilated regions of the lateral intercellular space (LIS), likely representing exit sites where Au-Fc accumulates en route to the blood. Before weaning, the jejunum functions primarily in IgG transport and exhibits unusual properties: clathrin-rich regions near/at the basolateral LIS and multivesicular bodies (MVBs) expressing early endosomal markers. To address whether these features are related to IgG transport, we examined LIS and endocytic/transcytotic structures from neonatal and weaned animals. Weaned samples showed less LIS-associated clathrin. MVBs labeled with late endosomal/lysosomal markers were smaller than their neonatal counterparts but contained 10 times more internal compartments. These results are consistent with hypotheses that clathrin-rich basolateral regions in neonatal jejunum are involved in IgG exocytosis and that MVBs function in IgG transport while FcRn is expressed but switch to degradative functions after weaning, when the jejunum does not express FcRn or transport IgG.     

MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the beta(2)-microglobulin (beta(2)m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical Fc gamma Rs: Fc gamma RI, Fc gamma RII, and Fc gamma RIII.     

FcRn: the neonatal Fc receptor comes of age

The neonatal Fc receptor for IgG (FcRn) has been well characterized in the transfer of passive humoral immunity from a mother to her fetus. In addition, throughout life, FcRn protects IgG from degradation, thereby explaining the long half-life of this class of antibody in the serum. In recent years, it has become clear that FcRn is expressed in various sites in adults, where its potential function is now beginning to emerge. In addition, recent studies have examined the interaction between FcRn and the Fc portion of IgG with the aim of either improving the serum half-life of therapeutic monoclonal antibodies or reducing the half-life of pathogenic antibodies. This Review summarizes these two areas of FcRn biology.     

The effect of the neonatal Fc receptor on human IgG biodistribution in mice

The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG. Recombinant therapeutic antibodies are typically composed of human or humanized sequences, and their biodistribution, or tissue distribution, is often studied in murine models, although, the effect of FcRn on tissue distribution of human IgG in rodents has not been investigated. In this report, an ¹²⁵I-labeled human IgG1 antibody was studied in both wild type C57BL/6 (WT) and FcRn knockout (KO) mice. Total radioactivity in both plasma and tissues (0–96hr post-dose) was measured by gamma-counting. Plasma exposure of human IgG1 were significantly lower in FcRn KO mice, which is consistent with the primary function of FcRn. Differences in biodistribution of human IgG to selected tissues were also observed. Among the tissue examined, the fat, skin and muscle showed a decrease in tissue-to-blood (T/B) exposure ratio of human IgG1 in FcRn KO mice comparing to the WT mice, while the liver, spleen, kidney, and lung showed an increase in the T/B exposure ratio in FcRn KO mice. A time-dependent change in the T/B ratios of human IgG1 was also observed for many tissues in FcRn KO mice. These results suggest that, in addition to its role in IgG elimination, FcRn may also play a role in antibody biodistribution.     

The effect of the neonatal Fc receptor on human IgG biodistribution in mice

The neonatal Fc receptor (FcRn) plays a pivotal role in IgG homeostasis, i.e., it salvages IgG antibodies from lysosomal degradation following fluid-phase pinocytosis, thus preventing rapid systemic elimination of IgG. Recombinant therapeutic antibodies are typically composed of human or humanized sequences, and their biodistribution, or tissue distribution, is often studied in murine models, although, the effect of FcRn on tissue distribution of human IgG in rodents has not been investigated. In this report, an ¹²⁵I-labeled human IgG1 antibody was studied in both wild type C57BL/6 (WT) and FcRn knockout (KO) mice. Total radioactivity in both plasma and tissues (0–96hr post-dose) was measured by gamma-counting. Plasma exposure of human IgG1 were significantly lower in FcRn KO mice, which is consistent with the primary function of FcRn. Differences in biodistribution of human IgG to selected tissues were also observed. Among the tissue examined, the fat, skin and muscle showed a decrease in tissue-to-blood (T/B) exposure ratio of human IgG1 in FcRn KO mice comparing to the WT mice, while the liver, spleen, kidney, and lung showed an increase in the T/B exposure ratio in FcRn KO mice. A time-dependent change in the T/B ratios of human IgG1 was also observed for many tissues in FcRn KO mice. These results suggest that, in addition to its role in IgG elimination, FcRn may also play a role in antibody biodistribution.     

Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells

The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.     

Neonatal Fc receptor expression in macrophages is indispensable for IgG homeostasis

ABSTRACT

The maintenance of the homeostasis of immunoglobulin G (IgG) represents a fundamental aspect of humoral immunity that has direct relevance to the successful delivery of antibody-based therapeutics. The ubiquitously expressed neonatal Fc receptor (FcRn) salvages IgG from cellular degradation following pinocytic uptake into cells, conferring prolonged in vivo persistence on IgG. However, the cellular sites of FcRn function are poorly defined. Pinocytic uptake is a prerequisite for FcRn-mediated IgG salvage, prompting us to investigate the consequences of IgG uptake and catabolism by macrophages, which represent both abundant and highly pinocytic cells in the body. Site-specific deletion of FcRn to generate mice harboring FcRn-deficient macrophages results in IgG hypercatabolism and ~threefold reductions in serum IgG levels, whereas these effects were not observed in mice that lack functional FcRn in B cells and dendritic cells. Consistent with the degradative activity of FcRn-deficient macrophages, depletion of these cells in FcRn-deficient mice leads to increased persistence and serum levels of IgG. These studies demonstrate a pivotal role for FcRn-mediated salvage in compensating for the high pinocytic and degradative activities of macrophages to maintain IgG homeostasis.     

NF-kappaB signaling regulates functional expression of the MHC class I-related neonatal Fc receptor for IgG via intronic binding sequences

The neonatal Fc receptor for IgG (FcRn) functions to transport maternal IgG to a fetus or newborn and to protect IgG from degradation. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown. Stimulation of intestinal epithelial cell lines, macrophage-like THP-1, and freshly isolated human monocytes with the cytokine TNF-alpha rapidly up-regulated FcRn gene expression. In addition, the TLR ligands LPS and CpG oligodeoxynucleotide enhanced the level of FcRn expression in THP-1 and monocytes. Treatment of TNF-stimulated THP-1 cells with the NF-kappaB-specific inhibitor or overexpression of a dominant negative mutant inhibitory NF-kappaB (IkappaBalpha; S32A/S36A) resulted in down-regulation of FcRn expression. By using chromatin immunoprecipitation we identified three NF-kappaB binding sequences within introns 2 and 4 of the human FcRn gene. An EMSA confirmed the p50/p50 and/or p65/p50 complex (s) bound to intron 2- or 4-derived oligonucleotides containing putative NF-kappaB binding sequences, respectively. The intronic NF-kappaB sequences in combination with the promoter or alone regulated the expression of a luciferase reporter gene in response to TNF-alpha stimulation or overexpression of NF-kappaB p65 and p50. DNA looping interactions potentially occurred after the stimulation between intronic NF-kappaB sequences and the FcRn promoter as shown by a chromosome conformation capture assay. Finally, TNF-alpha stimulations enhanced IgG transport across an intestinal Caco-2 epithelial monolayer. Together, these data provide the first evidence that NF-kappaB signaling via intronic sequences regulates FcRn expression and function.     

Expression of the neonatal Fc receptor (FcRn) at the blood-brain barrier

The blood-brain barrier (BBB) restricts transport of immunoglobulin G (IgG) in the blood to brain direction. However, IgG undergoes rapid efflux in the brain to blood direction via reverse transcytosis across the BBB after direct intracerebral injection. This BBB IgG transport system has the characteristics of an Fc receptor (FcR), but there is no molecular information on the putative BBB FcR. The present study uses confocal microscopy and an antibody to the rat neonatal FcR (FcRn), and demonstrates the expression of the FcRn at the brain microvasculature and choroid plexus epithelium. Co-localization with the Glut1 glucose transporter indicates the brain microvascular FcRn is expressed in the capillary endothelium. The capillary endothelial FcRn may mediate the 'reverse transcytosis' of IgG in the brain to blood direction.     

Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism

The neonatal FcR (FcRn) is a receptor that protects IgG from catabolism and is important in maintaining high serum Ab levels. A major site of expression of FcRn is vascular endothelial cells where FcRn functions to extend the serum persistence of IgG by recycling internalized IgG back to the surface. Because FcRn is expressed in other tissues, it is unclear whether endothelial cells are the only site of IgG protection. In this study, we used FcRn-deficient mice and specific antiserum to determine the tissue distribution of FcRn in the adult mouse. In addition to its expression in the vascular endothelium of several organs, we found FcRn to be highly expressed in bone marrow-derived cells and professional APCs in different tissues. Experiments using bone marrow chimeras showed that FcRn expression in these cells acted to significantly extend the half-life of serum IgG indicating that in addition to the vascular endothelium, bone marrow-derived phagocytic cells are a major site of IgG homeostasis.     

Quantitative cumulative biodistribution of antibodies in mice: Effect of modulating binding affinity to the neonatal Fc receptor

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn’s role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn’s role in antibody PK and catabolism at the tissue level.     

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (K_D) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.     

Characterization and screening of IgG binding to the neonatal Fc receptor

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies.

In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI).

Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities.

Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding.     

Characterization and screening of IgG binding to the neonatal Fc receptor

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from degradation and increases the serum half-life of IgG, thereby contributing to a higher concentration of IgG in the serum. Because altered FcRn binding may result in a reduced or prolonged half-life of IgG molecules, it is advisable to characterize Fc receptor binding of therapeutic antibody lead candidates prior to the start of pre-clinical and clinical studies. In this study, we characterized the interactions between FcRn of different species (human, cynomolgus monkey, mouse and rat) and nine IgG molecules from different species and isotypes with common variable heavy (VH) and variable light chain (VL) domains. Binding was analyzed at acidic and neutral pH using surface plasmon resonance (SPR) and biolayer interferometry (BLI). Furthermore, we transferred the well-accepted, but low throughput SPR-based method for FcRn binding characterization to the BLI-based Octet platform to enable a higher sample throughput allowing the characterization of FcRn binding already during early drug discovery phase. We showed that the BLI-based approach is fit-for-purpose and capable of discriminating between IgG molecules with significant differences in FcRn binding affinities. Using this high-throughput approach we investigated FcRn binding of 36 IgG molecules that represented all VH/VL region combinations available in the fully human, recombinant antibody library Ylanthia®. Our results clearly showed normal FcRn binding profiles for all samples. Hence, the variations among the framework parts, complementarity-determining region (CDR) 1 and CDR2 of the fragment antigen binding (Fab) domain did not significantly change FcRn binding.     

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (K_D) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics.     

Tissue expression profile of human neonatal Fc receptor (FcRn) in Tg32 transgenic mice

The neonatal Fc receptor (FcRn) is a homeostatic receptor responsible for prolonging immunoglobulin G (IgG) half-life by protecting it from lysosomal degradation and recycling it to systemic circulation. Tissue-specific FcRn expression is a critical parameter in physiologically-based pharmacokinetic (PBPK) modeling for translational pharmacokinetics of Fc-containing biotherapeutics. Using online peptide immuno-affinity chromatography coupled with high resolution mass spectrometry, we established a quantitative FcRn tissue protein expression profile in human FcRn (hFcRn) transgenic mice, Tg32 homozygous and hemizygous strains. The concentration of hFcRn across 14 tissues ranged from 3.5 to 111.2 pmole per gram of tissue. Our hFcRn quantification data from Tg32 mice will enable a more refined PBPK model to improve the accuracy of human PK predictions for Fc-containing biotherapeutics.     

The MHC class II-associated invariant chain interacts with the neonatal Fc gamma receptor and modulates its trafficking to endosomal/lysosomal compartments

The neonatal Fc receptor for IgG (FcRn) transfers maternal IgG to the offspring and protects IgG from degradation. The FcRn resides in an acidic intracellular compartment, allowing it to bind IgG. In this study, we found the association of FcRn and invariant chain (Ii). The interaction was initiated within the endoplasmic reticulum by Ii binding to either the FcRn H chain alone or FcRn H chain-beta(2)-microglobulin complex and appeared to be maintained throughout the endocytic pathway. The CLIP in Ii was not required for FcRn-Ii association. The interaction was also detected in IFN-gamma-treated THP-1, epithelial and endothelial cells, and immature mouse DCs. A truncated FcRn without the cytoplasmic tail was unable to traffic to early endosomes; however, its location in early endosomes was restored by Ii expression. FcRn was also detected in the late endosome/lysosome only in the presence of Ii or on exposure to IFN-gamma. In immature human or mouse DCs, FcRn was barely detected in the late endosome/lysosome in the absence of Ii. Furthermore, the cytoplasmic tail of Ii conferred tailless FcRn to route to both the early endosome and late endosome/lysosome in a hybrid molecule. Because the FcRn is expressed in macrophages and DCs or epithelial and endothelial cells where Ii is induced under inflammation and infection, these results reveal the complexity of FcRn trafficking in which Ii is capable of expanding the boundary of FcRn trafficking. Taken together, the intracellular trafficking of FcRn is regulated by its intrinsic sorting information and/or an interaction with Ii chain.     

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (K_D) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG''s variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG''s serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.     

Crystal structure at 2.8 A of an FcRn/heterodimeric Fc complex: mechanism of pH-dependent binding

The neonatal Fc receptor (FcRn) transports immunoglobulin G (IgG) across epithelia, binding IgG in acidic vesicles (pH < or = 6.5) and releasing IgG in the blood at pH 7.4. Well-ordered FcRn/Fc crystals are prevented by the formation of "oligomeric ribbons" of FcRn dimers bridged by Fc homodimers, thus we crystallized a 1:1 complex between rat FcRn and a heterodimeric Fc containing only one FcRn binding site. The 2.8 A complex structure demonstrates that FcRn uses its alpha2 and beta2-microglobulin domains and carbohydrate to interact with the Fc C(gamma)2-C(gamma)3 interface. The structure reveals conformational changes in Fc and three titratable salt bridges that confer pH-dependent binding, and can be used to guide rational design of therapeutic IgGs with longer serum half-lives.