Tissue expression profile of human neonatal Fc receptor (FcRn) in Tg32 transgenic mice.
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ABSTRACT:
SUBMITTER: Fan YY
PROVIDER: S-EPMC4968098 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Antibodies are essential components of an adaptive immune response. Immunoglobulin G (IgG) is the most common type of antibody found in circulation and extracellular fluids. Although IgG alone can directly protect the body from infection through the activities of its antigen binding region, the majority of IgG immune functions are mediated via proteins and receptors expressed by specialized cell subsets that bind to the fragment crystallizable (Fc) region of IgG. Fc gamma (γ) receptors (FcγR) belong to a broad family of proteins that presently include classical membrane-bound surface receptors as well as atypical intracellular receptors and cytoplasmic glycoproteins. Among the atypical FcγRs, the neonatal Fc receptor (FcRn) has increasingly gained notoriety given its intimate influence on IgG biology and its ability to also bind to albumin. FcRn functions as a recycling or transcytosis receptor that is responsible for maintaining IgG and albumin in the circulation, and bidirectionally transporting these two ligands across polarized cellular barriers. More recently, it has been appreciated that FcRn acts as an immune receptor by interacting with and facilitating antigen presentation of peptides derived from IgG immune complexes (IC). Here we review FcRn biology and focus on newer advances including how emerging FcRn-targeted therapies may affect the immune responses to IgG and IgG IC.
| S-EPMC6636548 | biostudies-literature
Project description:The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits--having one extra copy of the FcRn when hemizygous and two extra copies when homozygous--showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies.
| S-EPMC3256154 | biostudies-literature
Project description:BACKGROUND AND PURPOSE:Pemphigus and pemphigoid diseases are characterized and caused predominantly by IgG autoantibodies targeting structural proteins of the skin. Their current treatment relies on general and prolonged immunosuppression that causes severe adverse events, including death. Hence, novel safe and more effective treatments are urgently needed. Due to its' physiological functions, the neonatal Fc receptor (FcRn) has emerged as a potential therapeutic target for pemphigus and pemphigoid, primarily because IgG is protected from proteolysis after uptake into endothelial cells. Thus, blockade of FcRn would reduce circulating autoantibody concentrations. However, long-term effects of pharmacological FcRn inhibition in therapeutic settings of autoimmune diseases are unknown. EXPERIMENTAL APPROACH:Therapeutic effects of FcRn blockade were investigated in a murine model of the prototypical autoantibody-mediated pemphigoid disease, epidermolysis bullosa acquisita (EBA). B6.SJL-H2s C3c/1CyJ mice with clinically active disease were randomized to receive either an anti-FcRn monoclonal antibody (4470) or an isotype control over 4 weeks. KEY RESULTS:While clinical disease continued to worsen in isotype control-treated mice, overall disease severity continuously decreased in mice injected with 4470, leading to almost complete remission in over 25% of treated mice. These clinical findings were paralleled by a reduction of autoantibody concentrations. Reduction of autoantibody concentrations, rather than modulating neutrophil activation, was responsible for the observed therapeutic effects. CONCLUSION AND IMPLICATIONS:The clinical efficacy of anti-FcRn treatment in this prototypical autoantibody-mediated disease encourages further development of anti-FcRn antibodies for clinical use in pemphigoid diseases and potentially in other autoantibody mediated diseases.
| S-EPMC7174883 | biostudies-literature
Project description:Quantitative real-time PCR and Western blot methods were developed to assess neonatal Fc-receptor (FcRn) mRNA and protein expression in human FcRn transgenic mice, Swiss Webster mice, and in select human tissues. Additionally, FcRn turnover was evaluated via pulse-chase. FcRn mRNA expression was significantly higher in transgenic mice when compared to mouse FcRn mRNA in Swiss Webster mice and it ranged from 184-fold higher in the kidney to 109,000-fold higher in the skin. FcRn protein expression was found to be 13-fold lower in kidney to 5.6-fold higher in lung obtained from transgenic mice compared to FcRn protein expression in lung samples obtained from Swiss Webster mice. FcRn protein expression in human liver and small intestine tissues matched more closely with FcRn expression in Swiss Webster mice but were significantly lower when compared to values found from Swiss Webster and transgenic mice. Although FcRn mRNA expression correlated significantly with protein expression (p < 0.0005), the correlation coefficient was only 0.113. As such, the measurement of FcRn protein may be preferred to FcRn mRNA for quantitative applications. Significant differences were found in FcRn expression in transgenic mice, Swiss Webster mice, and human tissues, which may have implications for the use of mouse models in the assessment of monoclonal antibody disposition, efficacy, and safety.
| S-EPMC6316262 | biostudies-literature
Project description:System-wide quantitative characterization of human neonatal Fc receptor (FcRn) properties is critical for understanding and predicting human PK (pharmacokinetics) as well as the distribution of mAbs and Fc-fusion proteins using PBPK (physiologically-based pharmacokinetic) modeling. To this end, tissue-specific FcRn expression and half-life are important model inputs. Herein, human FcRn tissue expression was measured by peptide immunoaffinity chromatography coupled with high-resolution mass spectrometry. FcRn concentrations across 14 human tissues ranged from low to 230 pmol per gram of tissue. Furthermore, the FcRn half-life was determined to be 11.1 h from a human stable isotope labelled leucine pulse labeling experiment. The spatial and temporal quantitative human FcRn data now promise to enable a refined PBPK model with improved accuracy of human PK predictions for Fc-containing biotherapeutics.
| S-EPMC6722552 | biostudies-literature
Project description:IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2β, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2β, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2β, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.
| S-EPMC4966839 | biostudies-literature
Project description:Despite the increasing importance of rabbit as an animal model in pharmacological studies like investigating placental transfer of therapeutic IgGs, little is known about the molecular interaction of the rabbit neonatal Fc receptor (FcRn) with rabbit and human IgG molecules. We analyzed the interactions of the rabbit and human FcRn with rabbit and human IgG isotypes using surface plasmon resonance assay. Similar to FcRn of other species, rabbit FcRn functions in pH-dependent manner, as it binds IgGs at pH 6.0, but no binding occurs at pH 7.4. We also showed that rabbit FcRn binds rabbit IgG and human IgG1 with nearly identical affinity, whereas it has stronger interactions with the other human IgG isotypes. The similar affinity of rabbit IgG and human IgG1 for rabbit FcRn was confirmed by in vitro FcRn-mediated recycling assay. These data verify that rabbit is an appropriate animal model for analyzing the pharmacokinetics of human therapeutic monoclonal antibodies.
| S-EPMC5619814 | biostudies-literature
Project description:Albumin is the most abundant protein in blood and plays a pivotal role as a multitransporter of a wide range of molecules such as fatty acids, metabolites, hormones, and toxins. In addition, it binds a variety of drugs. Its role as distributor is supported by its extraordinary serum half-life of 3 weeks. This is related to its size and binding to the cellular receptor FcRn, which rescues albumin from intracellular degradation. Furthermore, the long half-life has fostered a great and increasing interest in utilization of albumin as a carrier of protein therapeutics and chemical drugs. However, to fully understand how FcRn acts as a regulator of albumin homeostasis and to take advantage of the FcRn-albumin interaction in drug design, the interaction interface needs to be dissected. Here, we used a panel of monoclonal antibodies directed towards human FcRn in combination with site-directed mutagenesis and structural modeling to unmask the binding sites for albumin blocking antibodies and albumin on the receptor, which revealed that the interaction is not only strictly pH-dependent, but predominantly hydrophobic in nature. Specifically, we provide mechanistic evidence for a crucial role of a cluster of conserved tryptophan residues that expose a pH-sensitive loop of FcRn, and identify structural differences in proximity to these hot spot residues that explain divergent cross-species binding properties of FcRn. Our findings expand our knowledge of how FcRn is controlling albumin homeostasis at a molecular level, which will guide design and engineering of novel albumin variants with altered transport properties.
| S-EPMC4059163 | biostudies-literature
Project description:The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure.
| S-EPMC3836734 | biostudies-literature
Project description:A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.
| S-EPMC4036356 | biostudies-literature