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1.
Mice genetically engineered to express human FcRn are valuable models for the evaluation of therapeutic antibodies in the context of human FcRn in vivo. However, only limited clinical chemistry information on these mouse strains is available. Thus, we have compared 30 clinical chemical parameters of C57BL/6J wild-type mice, murine FcRn-knockout mice, and two human FcRn transgenic mouse strains expressing human FcRn in the absence of murine FcRn. Since FcRn-mediated recycling prevents albumin and IgG from intracellular degradation, significant differences for both proteins were observed in the murine FcRn-knockout mice. Mice lacking FcRn show lower IgG and albumin levels compared to wild-type mice. The most prominent differences in clinical chemical parameters can be explained by secondary effects of the altered albumin levels of murine FcRn-knockout mice on liver metabolism, as similar tendencies have been observed in analbuminemic Nagase rats and hypoalbuminemic human patients, showing an overall increased liver metabolism. Both human FcRn transgenic strains show clinical chemical parameters similar to those found for wild-type mice, with the exception of endogenous IgG levels, which are greatly reduced in these mice.  相似文献   

2.
Normal albumin loss from the plasma is thought to be minimized by a number of mechanisms, including charge repulsion with the capillary wall and an intracellular rescue pathway involving the major histocompatibility complex-related Fc receptor (FcRn)-mediated mechanism. This study investigates how these factors may influence the mechanism of hypoalbuminemia. Hypoalbuminemia in rats was induced by treatment with puromycin aminonucleoside (PA). To test the effects of PA on capillary wall permeability, plasma elimination rates were determined for tritium-labeled tracers of different-sized Ficolls, negatively charged Ficolls, and (14)C-labeled tracer of albumin in control and PA-treated Sprague-Dawley rats. Urinary excretion and tissue uptake were also measured. Hypoalbuminemia was also examined in two strains of FcRn-deficient mice: beta(2)-microglobulin (beta(2)M) knockout (KO) mice and FcRn alpha-chain KO mice. The excretion rates of albumin and albumin-derived fragments were measured. PA-induced hypoalbuminemia was associated with a 2.5-fold increase in the plasma elimination rate of albumin. This increase could be completely accounted for by the increase in urinary albumin excretion. Changes in the permeability of the capillary wall were not apparent, inasmuch as there was no comparable increase in the plasma elimination rate of 36- to 85-A Ficoll or negatively charged 50- to 80-A Ficoll. In contrast, hypoalbuminemic states in beta(2)M and FcRn KO mice were associated with decreases in excretion of albumin and albumin-derived fragments. This demonstrates that the mechanism of hypoalbuminemia consists of at least two distinct forms: one specifically associated with the renal handling of albumin and the other mediated by systemic processes.  相似文献   

3.
The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.  相似文献   

4.
Engineered human IgG antibodies with longer serum half-lives in primates   总被引:3,自引:0,他引:3  
The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.  相似文献   

5.
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.  相似文献   

6.
《MABS-AUSTIN》2013,5(2):502-508
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.  相似文献   

7.
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.  相似文献   

8.
9.
The major histocompatibility complex class I-related neonatal Fc receptor, FcRn, assembles as a heterodimer consisting of a heavy chain and beta(2)-microglobulin (beta(2)m), which is essential for FcRn function. We observed that, in Madin-Darby canine kidney (MDCK) cells, the function of human FcRn in mediating the bidirectional transport of IgG was significantly increased upon co-expression of the human isoform of beta(2)m. In MDCK cells, the presence of human beta(2)m endowed upon human FcRn an enhanced ability to exit the endoplasmic reticulum and acquire mature carbohydrate side-chain modifications at steady state, a faster kinetics of maturation, and augmented localization at the cell surface as a mature glycoprotein able to bind IgG. Although human FcRn with immature carbohydrate side-chain modifications was capable of exhibiting pH-dependent binding of IgG, only human FcRn with mature carbohydrate side-chain modifications was detected on the cell surface. These results show that human FcRn travels to the cell surface via the normal secretory pathway and that the appropriate expression and function of human FcRn in MDCK cells depends upon the co-expression of human beta(2)m.  相似文献   

10.
An engineered human IgG1 antibody with longer serum half-life   总被引:1,自引:0,他引:1  
The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was approximately 2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.  相似文献   

11.
The neonatal FcR (FcRn) consists of an MHC class I-like H chain in noncovalent association with beta(2)-microglobulin (beta(2)m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the beta(2)m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and beta(2)m, CNX bound to the FcRn H chain before the FcRn H chain association with beta(2)m. However, calreticulin only bound to the FcRn H chain-beta(2)m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for beta(2)m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.  相似文献   

12.
FcRn is unique among immunoglobulin G (IgG) Fc receptors in that it is structurally closely related to major histocompatibility complex class I molecules and likewise consists of an alpha-chain and beta2-microglobulin. Crystallographic data show that rat FcRn alpha-chain/beta2m heterodimers can further dimerize via ionic interactions and a carbohydrate handshake. Intriguingly, however, no dimers are found in crystals of human FcRn, probably because the charged amino acids and the carbohydrate implicated in dimerization of rat FcRn are not conserved. Here, we show that although a secreted soluble form of human FcRn does not dimerize, the membrane-anchored receptor can form both non-covalent and covalent dimers. Furthermore, dimerization of human FcRn occurs in the absence of its ligand, IgG.  相似文献   

13.
《MABS-AUSTIN》2013,5(2):331-343
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 (KD) 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.  相似文献   

14.
The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, is active in phagocytosis and delivers antigen for presentation. We have previously shown that transgenic (tg) mice that have been created to overexpress bovine FcRn (bFcRn) demonstrate increased half-life of mouse IgG, significantly increased antigen-specific IgG in serum and augmented expansion of antigen-specific B cells and plasma cells after immunization. One of the interesting questions surrounding this enhanced immune response is whether these tg mice could effectively induce immune response to weakly immunogenic antigens. To address this question, we immunized these bFcRn tg mice with a conserved hemagglutinin subunit 2 (HA2)-based synthetic peptide that was recently found to be effectively targeted by neutralizing antibodies. Using an ELISA system, we found that, whereas wild-type mice showed a weak immune response and developed only a de minimis amount of antibody against the epitope, FcRn overexpressing animals mounted a robust reaction expressed in specific antibody titers on day 28 that continued to rise through day 50. Consistent with our previous data, the enhanced immune response resulting from the FcRn overexpression was also associated with a substantial increase in the number of spleen derived B cells, dendritic cells, granulocytes and plasma cells. Based on this evidence, we propose that tg mice that overexpress bFcRn offer major advantages in monoclonal antibody production because the tg mice would allow the generation of antibodies (hybridomas) to weakly immunogenic antigens that otherwise would be difficult or even impossible to make.Key words: neonatal Fc receptor (FcRn), transgenic mouse, immunogenicity, monoclonal antibody, influenza  相似文献   

15.
mAb HC-10 loses its reactivity with HLA class I (HLA-I) H chain (HC) following its association with beta(2)-microglobulin (beta(2)m). Furthermore, the HC-10 defined epitope appears to be involved in the pathogenesis of spondyloarthropathies, because HC-10 reduced their incidence in HLA-B27(+)beta(2)m degrees /MHC class II knockout mice. This study has characterized the determinant recognized by HC-10. Panning of a phage display peptide library with HC-10 resulted in isolation of the motif PxxWDR, which could be aligned with P57, W60, D61, and R62 of the first domain of the HLA-I HC allospecificities reactive with HC-10. The (55)EGPEYWDR(N/E)T(64) (p-1) is the shortest motif-bearing peptide that reacts with HC-10 and inhibits its binding to soluble HLA-B7 HC, irrespective of whether N (p-1a) or E (p-1b) is present at position 63. By contrast, HC-10 did not react with six additional peptides, each bearing motif amino acid substitutions present in HC-10-not-reactive HLA-I allospecificities. The p-1-derived Qp-1, synthesized with the additional conserved Q54, which displays the highest in vitro reactivity with HC-10, was the only one to induce in mice IgG resembling HC-10 in their fine specificity. Mapping of the HC-10-defined determinant suggests that the lack of mAb reactivity with beta(2)m-associated HLA-I HC is caused by blocking by the peptide in the groove of beta(2)m-associated HLA-I HC, though a role of HC conformational changes following its association with beta(2)m cannot be excluded. This information contributes to our understanding of the molecular basis of the antigenic profiles of beta(2)m-free and beta(2)m-associated HLA-I HC and may serve to develop active specific immunotherapy of spondyloarthropathies.  相似文献   

16.
《MABS-AUSTIN》2013,5(2):173-180
The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, is active in phagocytosis and delivers antigen for presentation. We have previously shown that transgenic (tg) mice that have been created to overexpress bovine FcRn (bFcRn) demonstrate increased half-life of mouse IgG, significantly increased antigen-specific IgG in serum and augmented expansion of antigen-specific B cells and plasma cells after immunization. One of the interesting questions surrounding this enhanced immune response is whether these tg mice could effectively induce immune response to weakly immunogenic antigens. To address this question, we immunized these bFcRn tg mice with a conserved hemagglutinin subunit 2 (HA2)-based synthetic peptide that was recently found to be effectively targeted by neutralizing antibodies. Using an ELISA system, we found that, whereas wild-type mice showed a weak immune response and developed only a de minimis amount of antibody against the epitope, FcRn over-expressing animals mounted a robust reaction expressed in specific antibody titers on day 28 that continued to rise through day 50. Consistent with our previous data, the enhanced immune response resulting from the FcRn overexpression was also associated with a substantial increase in the number of spleen derived B cells, dendritic cells, granulocytes and plasma cells. Based on this evidence, we propose that tg mice that overexpress bFcRn offer major advantages in monoclonal antibody production because the tg mice would allow the generation of antibodies (hybridomas) to weakly immunogenic antigens that otherwise would be difficult or even impossible to make.  相似文献   

17.
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 (KD) 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.  相似文献   

18.
The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule that regulates the half-life of IgG and albumin. In addition, FcRn directs the transport of IgG across both mucosal epithelium and placenta and also enhances phagocytosis in neutrophils. This new knowledge gives incentives for the design of IgG and albumin-based diagnostics and therapeutics. To study FcRn in vitro and to select and characterize FcRn binders, large quantities of soluble human FcRn are needed. In this report, we explored the impact of two free cysteine residues (C48 and C251) of the FcRn heavy chain on the overall structure and function of soluble human FcRn and described an improved bacterial production strategy based on removal of these residues, yielding approximately 70 mg.L(-1) of fermentation of refolded soluble human FcRn. The structural and functional integrity was proved by CD, surface plasmon resonance and MALDI-TOF peptide mapping analyses. The strategy may generally be translated to the large-scale production of other major histocompatibility complex class I-related molecules with nonfunctional unpaired cysteine residues. Furthermore, the anti-FcRn response in goats immunized with the FcRn heavy chain alone was analyzed following affinity purification on heavy chain-coupled Sepharose. Importantly, purified antibodies blocked the binding of both ligands to soluble human FcRn and were thus directed to both binding sites. This implies that the FcRn heavy chain, without prior assembly with human beta2-microglobulin, contains the relevant epitopes found in soluble human FcRn, and is therefore sufficient to obtain binders to either ligand-binding site. This finding will greatly facilitate the selection and characterization of such binders.  相似文献   

19.
Albumin binding to FcRn: distinct from the FcRn-IgG interaction   总被引:2,自引:0,他引:2  
The MHC-related Fc receptor for IgG (FcRn) protects albumin and IgG from degradation by binding both proteins with high affinity at low pH in the acid endosome and diverting both from a lysosomal pathway, returning them to the extracellular compartment. Immunoblotting and surface plasmon resonance studies show that both IgG and albumin bind noncooperatively to distinct sites on FcRn, that the affinity of FcRn for albumin decreases approximately 200-fold from acidic to neutral pH, and that the FcRn-albumin interaction shows rapid association and dissociation kinetics. Isothermal titration calorimetry shows that albumin binds FcRn with a 1:1 stoichiometry and the interaction has hydrophobic features as evidenced by a large positive change in entropy upon binding. Our results suggest that the FcRn-albumin interaction has unique features distinct from FcRn-IgG binding despite the overall similarity in the pH-dependent binding mechanism by which both ligands are protected from degradation.  相似文献   

20.
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.  相似文献   

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