IgG Transport Research Articles

Abstract 5341: Novel FcγR humanized mouse model enables assessment of PK/PD of therapeutic antibodies

Abstract Therapeutic antibodies have revolutionized the way we treat cancer. Enhanced activity of therapeutic IgG can be achieved by the modulation of Fc binding to Fcγ receptors (FcγR), which will consequently modulate the Fc-effector functions triggered upon crosslinking of target and effector cells by these antibodies. Assessment of therapeutic antibodies pharmacodynamic (PD) in preclinical models is challenging, as the FcγR are different from mice to human, as well as their expression pattern. FcRn is involved in the recycling and transport of IgG and is a key determinant of the pharmacokinetics (PK) of IgG. We report here a novel mouse model expressing human FcγR with a human-like expression pattern, in which human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB are expressed and replace their mouse orthologs. The model was intercrossed with the hSA/hFcRn mouse model (Viuff et al., 2016), which has shown to improve translatability of PK assessment of therapeutics with extended half-life via FcRn recycling and/or HSA binding. Investigation of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB expression on DC, monocytes, granulocytes, NK, T and B cells shows that expression pattern in hFcγR mice is consistent with their expression on human PBMC, with a few exceptions. These receptors are functional, as a human IgG1 targeting mouse CD20 induces approximately 50% of B-cell depletion in blood and liver, after a single iv administration in hFcγR mice. This depletion is Fc-mediated, as it is abrogated when hFcγR mice are treated with an anti-mouse CD20 human IgG1 with a Fc-null mutation. Assessment of antibody-dependent cell cytotoxicity (ADCC) ex vivo to investigate FcγR functionality in NK cells shows that Rituximab, an anti-human CD20 chimeric IgG1 with regular Fc, induces higher ADCC efficacy in NK cells from hFcγR than in NK cells from WT mice. Obinutuzumab, an anti-human CD20 featuring an optimized Fc portion to enhance binding to FcγRIII, shows higher tumor cell lysis than Rituximab in NK cells from hFcγR mice, suggesting this model enables ranking of antibodies. Moreover, assessment of activity of Fc-engineered anti-influenza IgG antibodies also shows that anti-Flu antibody with enhanced Fc binding outperforms the WT antibody, improves protective activity, and demonstrates effector function in influenza challenge model, following prophylactic treatment. Conversely, hFcγR mice treated with an Fc-null version of the anti-Flu antibody shows reduced protective activity and survival compared with the WT antibody. Therefore, the model reported here is a novel tool for testing efficacy of Fc-engineered therapeutic antibodies. We are currently investigating the PK profile of therapeutic antibodies in the hFcγR/hSA/hFcRn model. This novel tool is being improved to enable tolerability to human IgG1, biodistribution to the central nervous system, and flexibility of test of therapeutics through expression of human immune checkpoints. Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Rachel Courtois, Séverine Augier, Yacine Cherifi, Ariane Morel, Patricia Isnard-Petit, Kader Thiam. Novel FcγR humanized mouse model enables assessment of PK/PD of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5341.

295 Effects of a Supraphysiological Oxytocin Injection After Parturition on Blood-Milk-Barrier Closure and Igg Transfer into Milk in Dairy Cows

Abstract Oxytocin (OT) can affect the blood-milk-barrier integrity by modulating the closure of tight junctions. Earlier research demonstrated a transfer of circulating immunoglobulins (Ig) into milk upon OT application during an LPS-induced mastitis. The role of OT in colostrogenesis, i.e., IgG transfer into colostrum, is not fully understood. Our study aimed at retarding the postpartum closure of the blood-milk-barrier and concomitantly prolonging the transfer of IgG into colostrum of dairy cows by a supraphysiological OT injection. Multiparous Holstein cows (n = 28) were enrolled. Blood and milk samples were collected at the first 2 milkings after parturition and milk yields were recorded. Ten cows received 50 IU OT i.v. at 30 min after the first milking (OT+30), 9 cows were injected 50 IU OT i.v. at 120 min before the second milking (OT-120), and 9 cows remained untreated (CTRL). Cows were milked at 6.7 ± 0.7 h after parturition (first milking) and 11.5 ± 0.3 h later (second milking). Milk was analyzed for fat, protein, lactose and IgG, serum albumin (SA), and electrolytes (Na, K, Cl) concentrations and for lactate dehydrogenase (LDH) activity. Concentration of α-lactalbumin (α-LA) was measured in blood plasma. Data were evaluated using a mixed model (SAS) with group, milking number, and group x milking number as fixed effects, and cow as random subject. In all groups, concentrations of protein, IgG, SA, LDH in milk decreased from first to second milking, while lactose content in milk increased (P < 0.05). Milk yield and milk fat content did not differ between first and second milking (P > 0.05). Concentration of α-LA in plasma tended to decrease from first to second milking. Treatments did not affect changes in blood and milk from first to second milking. In conclusion, a single injection of supraphysiological OT either at 30 min after the first milking postpartum or 2 h before the second milking postpartum did not retard the closure of the blood-milk-barrier in dairy cows. Furthermore, OT application did not prolong the active transport of IgG into milk after the first milking.

Are high avidity antibodies to Plasmodium falciparum antigens preferentially transferred across the placenta of premature and term babies?

IntroductionTransplacental transport of maternal IgG via the neonatal Fc receptor (FcRn) provides babies with passive immunity. Several factors are reported to influence transport, including the avidity of antibodies (Abs) for their cognate antigens. Unfortunately, information on the role of antibody (Ab) avidity is limited. This study investigated if i) antibodies (Abs) with high avidity for 6 Plasmodium falciparum antigens and tetanus toxoid (TTx) were preferentially transferred to premature and term Cameroonian babies and ii) if Ab avidity was increased in babies whose mothers had placental malaria (PM), implicating the involvement of immune complexes. MethodsTotal IgG (mg/ml) and Abs to malarial antigens (AMA1, EBA-175, MSP1-42, MSP2, MSP3, DBL5 of VAR2CSA) and TTx were measured in paired mother-cord samples obtained from premature and term deliveries in Cameroon. Half the women had PM at delivery. Avidity Indices (AIs) were determined by treating antigen-bound-Abs with different molar concentrations of NH4SCN and calculating 50% endpoints. ResultsTotal IgG and antigen-specific Abs increased in cord blood with gestational age; however, AIs did not. AIs in paired maternal-cord blood samples were strongly associated for all antigens (r = 0.77–0.96). However, no significant different in AIs was found between paired mother-cord blood samples for any of the antigens (p values > 0.05). Similarly, Ab avidity was not increased in cord blood of babies whose mothers had PM or hypergammaglobulinemia. DiscussionOverall, there was no evidence that higher avidity Abs to any of the malarial antigens or TTx were preferentially transferred to Cameroonian babies.

МОРФОЛОГИЯ СИНЦИТИОТРОФОБЛАСТА ПЛАЦЕНТЫ В ТЕЧЕНИЕ ВСЕГО СРОКА НЕОСЛОЖНЕННОЙ БЕРЕМЕННОСТИ

The placenta is a fetal organ providing the interchange between mother and fetus. An important role in the regulation of local hemostasis belongs to the so-called brush border of syncytiotrophoblast villi, which is directly washed by maternal blood. Microvilli secrete placental alkaline phosphatase, which is a marker of transport processes and activates enzymes for many metabolic processes, participates in cell proliferation and tissue differentiation, carries out IgG transport to the fetus. The purpose of the work is to characterize the development of syncytiotrophoblast of the epithelium of the placenta villi at different dates of gestation during physiological pregnancy. To achieve the goal of the study, the tasks were set: 1. Study the dynamics of villi branching and the structure of the trophoblastic epithelium of villi from the stage of branched chorion to the end of pregnancy. 2. Determine the area of the trophoblastic epithelium of villi and study the immunohistochemical features of its main components using antibodies placental alkaline phosphatase, β – chorionic gonadotropin. 3. Study the dynamics of structural transformations and secretory activity of cyto- and syncytiotrophoblast at different gestational dates. Morphogenesis of syncytiotrophoblast of villous epithelium has been studied using histological, immunohistochemical and morphometric studies. The morphology of microvilli has been traced throughout the gestation period. The first forming microvilli were detected by 5 weeks; by 7 weeks, they were taller and flask-shaped; 8 weeks – had a branching structure; 9 weeks – non-branching structure, large and cylindrical; by the end of pregnancy (39-40 weeks) – rare, and there were no microvilli in the area of syncytio-capillary membranes. At 4-5 weeks, there is a weak degree of immunoexpression of β-hCG in the invasive cytotrophoblast and cytotrophoblastic epithelium and negative with the antibody against placental alkaline phosphatase. The area of the epithelium is 4,863.2 μm². At 6-7 weeks, the mean area of the villi is 19,705,5 μm². At 8-10 weeks, maximum epithelial area (6,370,7 μm²) and onset of placental alkaline phosphatase immunoexpression in the brush border area. In the period of 18-24 weeks, the area of villi decreases to 3,285,9 μm². At 39-40 weeks, terminal villi dominate with minimal values of the area of the trophoblastic epithelium, which is 786,2 μm². Syncytiocapillary membranes appear. A pronounced two-layer immunoexpression of placental alkaline phosphatase in the epithelium of all types of villi remains.

Transport of antibody into the skin is only partially dependent upon the neonatal Fc-receptor.

The dermis is the portal of entry for most vector-transmitted pathogens, making the host's immune response at this site critical in mitigating the magnitude of infection. For malaria, antibody-mediated neutralization of Plasmodium parasites in the dermis was recently demonstrated. However, surprisingly little is known about the mechanisms that govern antibody transport into the skin. Since the neonatal Fc receptor (FcRn) has been shown to transcytose IgG into various tissues, we sought to understand its contribution to IgG transport into the skin and antibody-mediated inhibition of Plasmodium parasites following mosquito bite inoculation. Using confocal imaging, we show that the transport of an anti-Langerin mAb into the skin occurs but is only partially reduced in mice lacking FcRn. To understand the relevance of FcRn in the context of malaria infection, we use the rodent parasite Plasmodium berghei and show that passively-administered anti-malarial antibody in FcRn deficient mice, does not reduce parasite burden to the same extent as previously observed in wildtype mice. Overall, our data suggest that FcRn plays a role in the transport of IgG into the skin but is not the major driver of IgG transport into this tissue. These findings have implications for the rational design of antibody-based therapeutics for malaria as well as other vector-transmitted pathogens.

Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.

Maternal antibiotic treatment during pregnancy attenuates the transport and absorption of maternal antibody IgG through TLR4 and TLR2 receptor.

Maternal antibody IgG, the main antibody in colostrum, plays an important role in neonates protection. Commensal microbiota is closely related to host antibody repertoire. However, there are few reports on how maternal gut microbiota affects maternal antibody IgG transfer. In the present study, we investigated the effects of altering the gut microbiota (treated with antibiotics during pregnancy) on maternal IgG transportation and offspring absorption and explored its underlying mechanisms. Results showed that antibiotic treatment during pregnancy significantly decreased maternal cecal microbial richness (Chao1 and Obesrved species) and diversity (Shannon and Simpson). Plasma metabolome enriched significant changes in the process of bile acid secretion pathway, and the concentration of deoxycholic acid, a secondary metabolite of microorganisms was lowered. Flow cytometry analysis indicated that antibiotic treatment promoted the number of B cells and abated the number of T, DC and M1 cells in intestinal lamina propria of dams. Surprisingly, the serum IgG level in antibiotic treated dams was significantly increased, while IgG contents in colostrum was decreased. Moreover, pregnancy antibiotic treatment in dams was reduced the expression of FcRn, TLR4 and TLR2 in breast of dams and in duodenum and jejunum of neonates. Furthermore, TLR4-/- and TLR2-/- knock-out mice showed a lower FcRn expression in breast of dams and in duodenum and jejunum of neonates. These findings suggest that maternal intestine bacteria may affect the maternal IgG transfer through regulating the breast TLR4 and TLR2 of dams.

Porcine Epidemic Diarrhea Virus nsp13 Protein Downregulates Neonatal Fc Receptor Expression by Causing Promoter Hypermethylation through the NF-κB Signaling Pathway.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic porcine enteric coronavirus that causes severe watery diarrhea and even death in piglets. The neonatal Fc receptor (FcRn) is the only transport receptor for IgG. FcRn expressed by intestinal epithelial cells can transport IgG from breast milk to piglets to provide immune protection. Previous studies have shown that viral infection affects FcRn expression. In this study, we showed for the first time, to our knowledge, that FcRn expression can be influenced by methyltransferases. In addition, we found that PEDV inhibited FcRn protein synthesis in porcine small intestinal epithelial cells postinfection. Then, we found that PEDV interfered with the transcription of genes through aberrant methylation modification of the FcRn promoter. DNA methyltransferase 3b (DNMT3b) has been implicated in this process. Using a series of PEDV structural and nonstructural protein (nsp) expression plasmids, we showed that nsp13 plays an important role in this aberrant methylation modification. PEDV nsp13 can affect the NF-κB canonical pathway and promote DNMT3b protein expression by facilitating p65 protein binding to chromatin. PEDV caused aberrant methylation of the FcRn promoter via DNMT3b. The same phenomenon was found in animal experiments with large white piglets. IgG transcytosis demonstrated that PEDV nsp13 can inhibit bidirectional IgG transport by FcRn. In addition, the core region of nsp13 (230-597 aa) is critical for FcRn inhibition. Taken together, to our knowledge, our findings revealed a novel immune escape mechanism of PEDV and shed new light on the design and development of vaccines and drugs.

Effect of Plasmodium Infection during Pregnancy on Passive Neonatal Immunity against Tetanus Toxoid and Rotavirus.

Passive immunity acquired through transplacental IgG transport is essential to protect infants against pathogens as childhood vaccination programs begins. Diarrhea caused by rotavirus and neonatal tetanus are common and potentially fatal childhood infections that can be prevented by transplacental IgG. However, it is not known whether maternal infections in pregnancy can reduce the transfer of these antibodies to the fetus. This study evaluated the effect of submicroscopic Plasmodium infection during pregnancy on the transfer of maternal IgG antibodies against rotavirus (anti-RV) and tetanus toxoid (anti-TT) to newborns of pregnant women residing in Puerto Libertador and Tierralta, Colombia. Expression of different immune mediators and levels of IgG against rotavirus and tetanus toxoid were quantified in pregnant women with and without Plasmodium infection during pregnancy. Submicroscopic infection at the time of delivery was associated with a cord-to-maternal ratio (CMR) >1 for anti-RV and <1 for anti-TT IgG, as well as with an increase in the expression of immune mediators of inflammation (IFN-γ), anti-inflammation (IL-10, TGF-β), and regulation (FoxP3, CTLA-4). When compared by species, these findings (CMR >1 for anti-RV and <1 for anti-TT IgG) were conserved in submicroscopic Plasmodium vivax infections at delivery. The impact of Plasmodium infections on neonatal susceptibility to other infections warrants further exploration.

Effects of a low- or high-frequency colostrum feeding protocol on immunoglobulin G absorption in newborn calves

Calves might experience an upper limit of IgG absorption from colostrum ingestion at birth, but it is not clear whether the total IgG mass fed in the first meal or feeding frequencies can saturate the IgG transport mechanism and therefore limit IgG absorption. The objective of this study was to determine whether different colostrum replacer (CR) feeding frequencies affect serum IgG levels or apparent efficiency of absorption (AEA) in neonatal calves. Male Holstein calves (n = 40) were separated from their dams immediately after parturition and randomly assigned to receive CR [12% of birth body weight (BW)], following either (1) a low-frequency (LF; n = 20) or (2) a high-frequency (HF; n = 20) feeding protocol. Low-frequency calves received 2 CR meals (8% and 4% birth BW within 1 h after birth and 12 h after first CR feeding, respectively), whereas HF calves received 3 CR meals (4% of BW for each meal; within 1 h after birth, 6, and 12 h after first CR feeding). The CR powder fed had a dry matter IgG concentration of 30% and an IgG concentration of 70.5 g/L when reconstituted. All CR was fed via esophageal tube within 1 h after birth. Calves were bottle-fed pasteurized milk (5% birth BW) at 24, 36, and 48 h after the first CR feeding. Blood was collected before first CR feeding and at the following intervals post-CR feeding: every 2 h until 18 h; every 3 h from 18 to 30 h; and every 6 h from 30 to 48 h after the first CR feeding. Serum IgG values at 24 h did not differ between LF and HF (25.79 ± 0.93 and 25.66 ± 0.88 g/L, respectively). In the first meal, calves fed LF ingested a higher total IgG mass than HF (257.98 ± 4.16 g and 126.72 ± 4.05 g, respectively); however, AEA at 24 h did not differ for calves fed HF or LF (27.68 ± 1.16% and 27.63 ± 1.26%, respectively). The IgG area under the curve (AUC) at 24 h was greater for calves fed LF than HF (443.13 ± 15.17 and 379.59 ± 13.99 g of IgG/L × h, respectively). Additionally, AUC at 6 h, 12 h, and 48 h were greater for calves fed LF than HF. These results indicate that, although LF calves had a greater AUC, HF calves were still able to absorb IgG in the second and third meal, allowing HF calves to achieve serum IgG levels similar to those of LF calves at 24 h. In addition, the provision of 3 meals at 70.5 g/L of IgG within the first 12 h of life did not result in added benefits to serum IgG or AEA levels.

Minimal Physiologically-based Pharmacokinetic Model to Investigate the Effect of Charge on the Pharmacokinetics of Humanized anti-HCV-E2 IgG Antibodies in Sprague-Dawley Rats.

To develop a minimal physiologically-based pharmacokinetic (mPBPK) model in quantifying the relationships between the charge and pharmacokinetics (PK) of therapeutic monoclonal IgG antibody (TMAb). PK data used in this study were native IgG and five humanized anti-HCVE2-IgG antibodies in rats. Different models that related the effect of charge on interstitial distribution, transcapillary transport, and cellular uptake for FcRn-mediated metabolism were tested. External validation was conducted to assess if the charge-parameter relationships derived from rats could be used to predict the PK of TMAbs in mice. The final mPBPK model was used to construct the relationships between the FcRn binding and charge on the PK of TMAbs. Increasing the isoelectric point (pI) of IgG was associated with higher interstitial space distribution and cellular uptake. The transcapillary transport of IgG from plasma to interstitial space remains constant with pI values below 7.96 and then increased linearly with pI. The model-based simulation results suggested that improving the FcRn binding affinity can overcome the problems of low plasma/interstitial space exposures associated with TMAbs with higher pI values by reducing the FcRn-mediated metabolism and hence increasing drug exposure in the interstitial space that has close contact with many solid tumors. The final mPBPK model was developed and used to construct complex quantitative relationships between the pI/FcRn binding affinity and PK of TMAbs and such relationships are useful to select the discovery of a "sweet spot" of designing future generation of TMAbs with optimal PK properties to achieve desirable plasma and tissue drug exposures.

Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

Persons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~ 10% increase), decreased IgG content in intracellular vesicles (~ 9% decrease), and a trend towards decreased IgG recycling (~ 55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~ 60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.

AB0020 ACPA ILLUSTRATING THE IMPACT OF IGG FAB-GLYCOSYLATION ON TRANSPLACENTAL TRANSFER OF ANTIBODIES AND THEIR BINDING TO THE NEONATAL FC-RECEPTOR (FCRN)

Background:Fc neonatal receptor (FcRn) is crucial for IgG half-life and transplacental transport. Different sites of IgG carry glycans which may affect binding to FcRn. While the effect of Fc-glycans has been investigated, the impact of Fab-glycosylation (~14% IgG) on IgG-FcRn interaction remains unclear. Anti-citrullinated protein antibodies (ACPA) of rheumatoid arthritis patients exhibit remarkably high Fab-glycosylation (~90%). This makes ACPA an ideal model to investigate how Fab-glycosylation influences IgG-FcRn interaction.Objectives:To investigate the potential impact of IgG Fab-glycosylation on IgG transplacental transfer and interaction with FcRn.Methods:To investigate transplacental transport of ACPA and total IgG, serum of ACPA-positive RA patients (mothers) as well as of healthy mothers and their respective newborns was analyzed. IgG Fab- and Fc-glycosylation was investigated with liquid chromatography and mass-spectrometry. Furthemore, ACPA monoclonal IgG were produced and glycoengineered to acquire several variants of the same monoclonal antibody differing only in their glycosylation profile. These glycovariants were then used to investigate the impact of Fab-glycans on the affinity of IgG for FcRn. Surface plasmon resonance (SPR) and affinity chromatography were implemented.Results:When measured in mothers’ serum and cord blood samples, Fab-glycosylation of IgG antibodies was ~20% lower in newborns compared to their mothers, which was observed for ACPA IgG, non-ACPA IgG in RA patients and total IgG of healthy controls (Figure 1). This may indicate that transplacental transfer of Fab-glycosylated antibodies is impaired. SPR results suggested that presence of Fab-glycans slightly lowered the affinity of IgG for FcRn. However, presence of Fab-glycans did not have a significant effect on the results of FcRn affinity chromatography. Together, these results suggest that Fab-glycans may impair association of IgG with FcRn, while dissociation likely stays intact.Conclusion:Our results suggest that Fab-glycans inhibit IgG-FcRn binding which negatively affects the transplacental transfer of Fab-glycosylated IgG. The impact of Fab-glycosylation on IgG half-life requires further investigation.Figure 1.Disclosure of Interests:Mikhail Volkov: None declared, Karin van Schie: None declared, Albert Bondt: None declared, Theresa Kissel: None declared, Maximilian Brinkhaus Grant/research support from: argenx, Arthur Bentlage: None declared, Carolien Koeleman: None declared, Steven de Taeye Grant/research support from: Genmab, Radboud Dolhain: None declared, Manfred Wuhrer: None declared, Rene Toes: None declared, Gestur Vidarsson: None declared, Diane van der Woude: None declared

FcRn Overexpression Expands Diversity of the Humoral Immune Response in bFcRn Transgenic Mice.

The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In this study we analyzed the diversity of the humoral immune response of bFcRn Tg mice, using a length distribution analysis (spectratyping) and next generation sequencing (NGS) of the immunoglobulin heavy chain variable regions. Our analysis showed that in response to immunization with ovalbumin or transfected cells that expressed a unique membrane protein, our Tg animals developed a more diverse plasma cell repertoire than controls, which manifested in greater numbers of different clones, and clusters with fewer highly expanded large clones, as identified by the variable region (CDR3) of the immunoglobulin heavy chain. The increased antibody diversity in Tg mice after immunization was observed at both IgM and IgG levels, indicating that the increased humoral immune diversity in Tg mice is due to a higher number of both activated, antigen-specific naïve and isotype switched B cells. We thus demonstrated that the BCR repertoire of the immunized bFcRn Tg animals is more diverse compared to wild type mice, which likely makes these Tg mice a better choice for monoclonal antibody production against challenging antigens, including the extracellular regions of cell membrane proteins.

FcRn, but not FcγRs, drives maternal-fetal transplacental transport of human IgG antibodies

The IgG Fc domain has the capacity to interact with diverse types of receptors, including the neonatal Fc receptor (FcRn) and Fcγ receptors (FcγRs), which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis, are mediated by FcγRs, which upon cross-linking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and nonoverlapping functional properties of FcRn and FcγRs, recent studies have suggested that FcγRs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of FcγRs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter FcγRIIIa or FcRn binding, half-life, or their ability to deplete target cells in FcγR/FcRn humanized mice. Modeling maternal-fetal transport in FcγR/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing FcγRIIIa binding did not result in enhanced maternal-fetal transport. These results argue against a role for FcγRs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to enhance only FcRn binding as a means to improve maternal-fetal transport of IgG.

Transmissible Gastroenteritis Virus Infection Up-Regulates FcRn Expression via Nucleocapsid Protein and Secretion of TGF-β in Porcine Intestinal Epithelial Cells.

Transmissible gastroenteritis virus (TGEV) is a porcine intestinal coronavirus that causes fatal severe watery diarrhea in piglets. The neonatal Fc receptor (FcRn) is the only IgG transport receptor, its expression on mucosal surfaces is triggered upon viral stimulation, which significantly enhances mucosal immunity. We utilized TGEV as a model pathogen to explore the role of FcRn in resisting viral invasion in overall intestinal mucosal immunity. TGEV induced FcRn expression by activating NF-κB signaling in porcine small intestinal epithelial (IPEC-J2) cells, however, the underlying mechanisms are unclear. First, using small interfering RNAs, we found that TGEV up-regulated FcRn expression via TLR3, TLR9 and RIG-I. Moreover, TGEV induced IL-1β, IL-6, IL-8, TGF-β, and TNF-α production. TGF-β-stimulated IPEC-J2 cells highly up-regulated FcRn expression, while treatment with a JNK-specific inhibitor down-regulated the expression. TGEV nucleocapsid (N) protein also enhanced FcRn promoter activity via the NF-κB signaling pathway and its central region (aa 128–252) was essential for FcRn activation. Additionally, N protein-mediated FcRn up-regulation promotes IgG transcytosis. Thus, TGEV N protein and TGF-β up-regulated FcRn expression, further clarifying the molecular mechanism of up-regulation of FcRn expression by TGEV.

Isolation and Identification of Porcine Epidemic Diarrhea Virus and Its Effect on Host Natural Immune Response.

Porcine epidemic diarrhea (PED) is a highly infectious intestinal disease caused by porcine epidemic diarrhea virus (PEDV). A PEDV strain was isolated from the piglet intestinal tract in Vero cells in Jiangsu Province, designated as the JS-A strain. PEDV was identified as the isolated virus by cytopathology, immunofluorescence assay, western blotting, transmission electron microscopy, and sequence analysis. The full-length genome of the JS-A isolate and the S gene were systematically analyzed, indicating that PEDV JS-A belongs to the G2a subtype, which is closely related to the prevalent PEDV in many countries and different from many current vaccines. Animal regression tests showed that piglets that are orally infected with the virus continue to develop diarrhea with yellowish and unpleasant odors. Further, piglets showed reduced food consumption and weight loss in the challenged group, while there were no abnormalities in the control group. In addition, Toll-like receptors (TLRs), RIG-I, and the downstream medium gene in the intestinal mucosa of newborn pigs infected with PEDV JS-A strain were studied. The neonatal Fc receptor (FcRn) was the only IgG transport receptor and protected IgG from degradation. Therefore, PEDV JS-A infection might inhibit FcRn expression by down-regulating TLRs and downstream signaling molecules. Taken together, isolation of the JS-A variant contributes to evolutionary analysis of the diarrhea virus. Further, the experimental infection model lays a foundation for further research related to vaccine development and the antiviral natural immune response of infected piglets, which helps us to better understand PEDV pathogenesis and immune mechanism.

Human cytomegalovirus evades antibody-mediated immunity through endoplasmic reticulum-associated degradation of the FcRn receptor

Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with β2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgG-FcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune surveillance.

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.

Neonatal Fc receptor (FcRn) – not only transporter of maternal IgG

The neonatal Fc receptor, (FcRn) is a transmembrane, heterodimeric glycoprotein with a structure similar to MHC class I molecules. In contrast to MHC class I antigens, FcRn does not bind to peptides (antigens) but interacts with the Fc fragment of IgG and albumin. The FcRn-IgG interaction as well as the FcRn-albumin interaction occur at acidic pH (optimally at pH 5.0-6.5) but not in physiological environment. These two ligands bind to distinct binding sites on the receptor molecule and by different mechanisms. Now, it is known that the expression of FcRn is not restricted to sites involved in the transport of maternal IgG, and this receptor is not responsible only for transfer the passive immunity from mother to the offspring. But FcRn has a much broader range of expression and function, throughout life and in many different cell types and tissues of humans and animals. This review summarizes the status of our knowledge on the expression, interaction with ligands and functions of the neonatal Fc receptor. This article shows also the possibilities of utilizing a current knowledge on FcRn for therapeutic purposes.