Skip Navigation


International Immunology Advance Access originally published online on February 15, 2006
International Immunology 2006 18(4):525-536; doi:10.1093/intimm/dxh393
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/4/525    most recent
dxh393v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Kacskovics, I.
Right arrow Articles by Hammarström, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kacskovics, I.
Right arrow Articles by Hammarström, L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

FcRn mediates elongated serum half-life of human IgG in cattle

Imre Kacskovics1, Zsuzsanna Kis1, Balázs Mayer1, Anthony P. West, Jr2, Noreen E. Tiangco2, Mulualem Tilahun3, László Cervenak4, Pamela J. Bjorkman2, Richard A. Goldsby3, Ottó Szenci5 and Lennart Hammarström6

1 Department of Physiology and Biochemistry, Faculty of Veterinary Science, Szent István University, Budapest, Hungary
2 Division of Biology 114-96 and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA
3 Department of Biology, Amherst College, Amherst, MA, USA
4 Research Group of Metabolism and Atherosclerosis, Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary
5 Clinics for Large Animals, Faculty of Veterinary Science, Szent István University, Üllo, Hungary
6 Division of Clinical Immunology, Karolinska Hospital, Huddinge, Sweden

Correspondence to: I. Kacskovics; E-mail: kacskovics.imre{at}aotk.szie.hu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
IgG has the longest survival time in the circulation of the Ig classes and the lowest fractional catabolic rate. The neonatal Fc receptor (FcRn) plays an important role in regulating these processes. Recently, we have cloned the bovine neonatal Fc receptor (bFcRn) alpha chain and detected its expression in various epithelial cells which are mediating IgG secretion. However, its function in IgG homeostasis has not been investigated. In the current study, we analyzed the binding affinity of bovine and human IgGs to bFcRn using surface plasmon resonance and by in vitro radioreceptor binding assays. As human IgG binds stronger to the bFcRn, than bovine IgG at pH 6, we subsequently analyzed its catabolism in normal and transchromosomic calves that produce human Igs. Pharmacokinetic studies showed that human IgG had ~33 days serum half-life both in normal and transchromosomic calves, which is more than two times longer than its bovine counterpart. We also demonstrate FcRn expression in endothelial cells and in the kidney which are supposed to be involved in IgG metabolism. These data suggest that bFcRn is involved in IgG homeostasis in cattle and furthermore, that the transchromosomic calves producing human Igs can effectively protect their human IgGs which have implications for successful large-scale production of therapeutic antibodies.

Keywords: antibody, clearance, interaction, therapeutic antibodies, transchromosomic cattle


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Human polyclonal antibodies can be used for a wide variety of therapeutic applications, including treatment of antibiotic-resistant infections, immune deficiencies and various autoimmune diseases. However, as they are available only from human donors who cannot be readily hyperimmunized, supply is limited. Transgenic mice carrying human Ig loci have previously been created (1–3) and are useful for the derivation of human mAbs antibodies. However, large-scale production of human antibodies cannot be gained from these animals. To address this need, transchromosomic calves carrying the human Ig heavy-chain and light-chain loci have recently been produced. These animals express diversified transcripts and human Igs have been detected in the blood (4). Since the majority of human therapeutic antibodies are of the IgG class, it is of importance to understand human IgG homeostasis in cattle.

The metabolism of IgG differs from those of the other classes of Igs, in that IgG has the longest survival time in the circulation and the lowest fractional catabolic rate (5). IgG elimination is likely dominated by affinity for the neonatal Fc receptor (FcRn), and the nature of and affinity for the specific target of the antibody (6, 7). However, other factors may contribute to the rate of antibody elimination, including the immunogenicity of the antibody (8), the degree and nature of the glycosylation of the antibody (9–11) and the susceptibility of the antibody to proteolysis (12).

The FcRn is a heterodimer that comprises a transmembrane {alpha} chain with structural homology to the extracellular domains of the {alpha} chain of MHC class I molecules, and a light chain consisting of beta 2-microglobulin (ß2m) (13). FcRn mediates both transcytosis of maternal IgG to the fetus or neonate and IgG homeostasis in adults (14). Evidence for the latter role initially derived from studies indicates an unusually short serum half-life of IgG in ß2m-deficient mice (15–17). This observation led to the generation of mutant mouse Fc fragments with enhanced binding to FcRn and increased serum persistence in mice (18), and in a more recent study a similar observation has been published with mutant human IgGs in rhesus monkeys (19). Earlier pharmacokinetic studies indicated that most plasma proteins, including IgG, were catabolized in close contact with the vascular space, which led to the hypothesis that the catabolic site for IgG and other proteins was most likely the vascular endothelium (5). Analyses in mice have proven that IgG homeostasis is indeed maintained by vascular endothelial cells (20). Interestingly, FcRn also binds albumin and prolongs its half-life in a concentration-dependent manner like is seen for IgG (21, 22).

In cattle, the serum IgG level is ~20 mg ml–1, and the two major IgG isotypes (IgG1, IgG2) are present in nearly equal amounts. The half-lives of IgG1 and IgG2 have been reported in several studies but the values are divergent between publications [reviewed in (23)]. However, the data indicate that they both fall in the range of 10–22 days (24, 25) with a slightly longer half-life for IgG2 (26, 27). It is worth mentioning, that in addition to vascular catabolism, a significant amount of IgG1 is secreted onto mucosal surfaces that may influence its apparent half-life in serum [reviewed in (28)]. The clearance rates of other bovine Ig classes, similar to other mammalian species, are much shorter, with half-lives of 4.8, 3.4 and 1.9 days for IgM, IgA and IgE, respectively (25).

In ruminants, the FcRn {alpha} chain has been cloned, characterized and its deduced amino acid sequence shows the highest similarity to human neonatal Fc receptor (hFcRn) among the non-primate species (29, 30). Although the presence of FcRn transcripts in multiple mucosal epithelial cells, which are considered to secrete IgG, has been confirmed (30, 31), its exact role in IgG transport has not been elucidated and even less is known about its function in IgG homeostasis.

In this study, we have analyzed the interaction of human and bovine IgG with bovine neonatal Fc receptor (bFcRn) by surface plasmon resonance (SPR), radioreceptor assays and the half-life of human IgG in normal and transchromosomic calves producing human Igs.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Expression and purification of the soluble bovine FcRn
The soluble bovine FcRn (sbFcRn) heavy-chain cDNA (116–982 bp; GenBank AF139106) was PCR amplified with Deep Vent polymerase (New England Biolab, Beverly, MA, USA) from a eucaryotic expression construct described before (29). The forward primer contains an EcoRI site, a ‘Kozak’ sequence (italic) and the coding region (underlined) (bFcRnBACU_for: 5'-ATC AGA ATT CCC TAT AAA TAT GCG GCT TCC CCG GCC T-3'); the reverse primer contains the gene-specific coding regions (underlined), a factor Xa site (italic), a 6xHis tag coding sequence, a stop codon and an EcoRI site (bFcRnBACU_rev: 5'-ATC AGA ATT CCT AAT GAT GAT GAT GAT GAT GAC GAC CTT CGA TCA GCT CCA CCG TGA GGG GCT-3'). The light-chain (bovine beta 2-microglobulin; bß2m) cDNA was PCR amplified (11–373 bp; GenBank X69084) with Deep Vent polymerase from a construct which was a kind gift from Shirley Ellis (Compton, UK) (32). The forward primer contains a BamHI site, a Kozak sequence (italic) and the coding region (underlined) (BB2MBACU_for: 5'-ATC AGG ATC CCC TAT AAA TAT GGC TCG CTT CGT GGC CTT-3'), the reverse primers contain a BamHI site with the coding region (BB2MBACU_rev: 5'-ATC AGG ATC CTG CTG CTT ACA GGT CTC GAT-3'). The sbFcRn and bß2m fragments were digested with EcoRI and BamHI, respectively, and ligated into pAcUW51 baculovirus transfer vector (BD Biosciences, San Diego, CA, USA). Clones harboring plasmid constructs with the desired orientation of the gene fragments were fully sequenced.

Recombinant bFcRn was purified from supernatants of baculovirus-infected insect cells (High-5; Invitrogen, Carlsbad, CA, USA), buffer exchanged to 50 mM phosphate at pH 8.0, followed by Ni-NTA chromatography (Ni-NTA superflow, Qiagen, Chatsworth, CA, USA). Protein from an imidazole elution was further purified by gel filtration chromatography and then analyzed on a 15% SDS–PAGE gel, followed by Coomassie blue staining.

SPR experiments
A Biacore 2000 biosensor system was used to assay the interaction of human and bovine IgGs with bFcRn and hFcRn (33). The biosensor system includes a chip with a dextran-coated gold surface to which one protein (the ‘ligand’) is covalently immobilized. Binding of an injected protein (the ‘analyte’) to the immobilized protein results in changes in the SPR that are proportional to the amount of bound protein and read out in real time as resonance units (RU). hFcRn and bFcRn were immobilized by amine coupling chemistry on seperate flowcells at similar coupling densities (hFcRn, 1700 RU; bFcRn, 1800 RU), and a third flowcell was mock coupled using buffer to serve as a blank. Serial dilutions of bovine and human IgGs (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were injected at room temperature in 50 mM sodium phosphate, pH 6.0, and 150 mM NaCl. Binding reactions were allowed to closely approach or to reach equilibrium and KDs were derived by non-linear regression analysis of plots of Req (the equilibrium binding response) versus the analyte concentration. Binding data were fit to a model with two classes of non-interacting binding sites as described for the analysis of FcRn with IgG (33, 34). This model assumes that there are two classes of IgG binding sites on the chip and derives macroscopic equilibrium dissociation constants (KD1 and KD2) and the precentage of the total response due to each class of binding sites (f1 and f2). The higher affinity binding constant (KD1), which reflects IgG being bound by two immobilized FcRn molecules (one by each heavy-chain Fc domain), is comparable to the affinities measured in cell binding assays involving membrane-bound FcRn (35, 36).

bFcRn stable-transfected cells
bFcRn heavy-chain stable-transfected bovine mammary epithelial cell line (MAC-T; a kind gift from A. Guidry, USDA, Beltsville, MD, USA) was generated as described previously (29). Briefly, a construct of the full length of bFcRn heavy-chain cDNA cloned into the pCI-neo eucaryotic expression vector (Promega Corp., Madison, WI, USA) was transfected into MAC-T cells using Ca-phosphate (37, 38). Cells were then selected with G418 (600 µg ml–1) and individual G418-resistant colonies were tested for the presence of the bFcRn by western blot using an affinity-purified specific antibody against an oligopeptide (CLEWKEPPSMRLKAR, amino acids 173–186) of bFcRn (30).

pH-dependent IgG binding and uptake of the transfected and untransfected cells were analyzed by single-point competitive binding assay according to the protocol of Story et al. (39). Briefly, bovine and human IgGs (Sigma–Aldrich, St Louis, MO, USA) were labeled with Na125I to a specific activity of ~10 Ci µmol–1 using Chloramine-T (Sigma–Aldrich). The cells expressing the bFcRn were first washed with DMEM, pH 6 or 7.5. Then, bovine-125I-IgG or human-125I-IgG in DMEM, pH 6.0 or 7.5 with or without unlabeled bovine, human IgG and chicken IgY (Sigma–Aldrich) in 1000 molar excess was added. The cells were allowed to bind and internalize IgG for 2 h at 37°C and then unbound ligand was removed with washes of chilled PBS, pH 6.0 or 7.5. Bound radioligand was measured in a gamma counter. Viability of the cells before and after the assay was checked by trypan blue exclusion assay.

Competitive binding assays
For the competitive binding assay, we measured the binding of a single concentration of radiolabeled human IgG in the presence of various concentrations of unlabeled human and bovine IgGs on transfected cells. The binding assay was performed as it was indicated above, except we performed the assay only at pH 6 with increasing amount (2-fold serial dilutions from 1000- to 0.06-fold molar excess) of unlabeled bovine and human IgG, as competitors. The cells were allowed to bind and take up IgG for 2 h at 37°C; then unbound ligand was removed with washes of chilled PBS (pH 6.0). Inhibitory concentration 50% (IC50) values were calculated using Prism for Windows, version 4.0 (GraphPad Software, San Diego, CA, USA).

FcRn detection in an endothelial cell line (bovine aortic endothelial cells), in tissue capillary endothelial cells and in the kidney
Reverse transcription–PCR.
RNA was isolated from primary bovine aortic endothelial cells (bAECs, Cambrex Bio Science, Walkersville, MD, USA) by TRIzol® reagent (Invitrogen). cDNA was synthesized using standard methods and was subsequently used in PCR with a primer pair specific for bFcRn (matching or complementary to bases 914–932 or 1028–1047). PCR products were analyzed by agarose gel electrophoresis.

Immunohistochemistry.
Intestinal, skin and kidney samples were collected at the slaughterhouse from healthy Holstein cattle. Samples were analyzed by immunohistochemistry as described previously (30). Briefly, the samples were treated with 1% H2O2 to inactivate endogenous peroxidases, and then blocked by Tris-buffered saline containing 5% BSA. Sections were subsequently incubated with affinity-purified anti-FcRn in 1% BSA and then with biotinylated goat anti-rabbit IgG. The secondary antibody was detected with Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and 3,3'-diaminobenzidine (Sigma Chemical Co., St Louis, MO, USA) was used as a color substrate.

In vivo studies of human IgG and chicken IgY half-lives in calves
Four Holstein calves (heifers), ~200 kg, were used to analyze the human IgG clearance. Following a pre-bleed, each calf was injected intravenously (i.v.) with 2 g (10 mg per body weight kilogram; BWkg) of human IgG (Gammonativ intended for intravenous use was a kind gift from Octapharma, Stockholm, Sweden) in 50 mg ml–1 saline solution and during the next 35 days, periodic blood samples were collected and evaluated for their content of human IgG.

A quantitative ELISA employing an unlabeled affinity-purified goat anti-human polyclonal antibody (goat anti-human IgG (H + L); Pierce Biotechnology Inc., Rockford, IL, USA) as capture reagent and an HRP-conjugated affinity-purified polyclonal goat anti-human IgG (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) as detecting reagent was used to evaluate plasma concentrations of human IgG during the course of the experiment. The peroxidase-conjugated antibody was detected using o-phenylenediamine (Fluka Chemie GmbH, Buchs, Switzerland) as the substrate. Samples were assayed in triplicate. Analysis of the mean human IgG concentration of the animals in the first 6.8 days was done by fitting the data to the two-compartmental model using WinNonLin Professional, version 4.1 (Pharsight, Mountain View, CA, USA). The individual elimination curves were used to obtain the pharmacokinetic parameters. The individual values were averaged, and the standard deviation was calculated.

Bovine IgG specific to the human IgG was detected by an ELISA assay employing the human IgG as capture reagent and after blocking (in 0.02 M Tris buffer containing 0.1% Tween 20 and 1mg ml–1 ovalbumin for 1 h at room temperature), incubating with a peroxidase-conjugated affinity-purified goat anti-bovine IgG (H + L) (Jackson ImmunoResearch Laboratories, Inc.) as a detecting reagent. Samples were assayed in triplicate.

In a control experiment, we injected 1 g (5 mg per BWkg) of chicken IgY which was purified from egg yolk (a kind gift from Vilmos Palya, CEVA Phylaxia, Budapest, Hungary) i.v. into two Holstein calves weighing ~200 kg and during the next 21 days periodic blood samples were collected. A quantitative ELISA employing a monoclonal anti-chicken IgY (Biodesign International, Saco, ME, USA) as capture reagent and an HRP-conjugated affinity-purified polyclonal rabbit anti-chicken IgY (Fc) (Biodesign International) as detecting reagent was used to evaluate plasma concentrations of chicken IgY during the course of the experiment. Samples were assayed in triplicate. Pharmacokinetic parameters were calculated by non-compartmental method, using the computer program WinNonLin Professional, version 4.1 (Pharsight). Serum concentration–time curve was calculated using the linear trapezoidal rule; however this was estimated by extrapolation from the apparent linear portion of the semi-logarithmic plot after the predicted steady-state situation (day 2) and before the change in the kinetics due to the onset of the immune response against the chicken IgY. The individual elimination curves were used to obtain the pharmacokinetic parameters. The individual values were averaged.

All experimental procedures were approved by the Animal Care and Ethics Committee of the Faculty of Veterinary Science, Szent István University and complied with the Hungarian Code of Practice for the Care and Use of Animals for Scientific Purposes.

In vivo studies of human IgG half-life in human artificial chromosome-transgenic cattle
Two calves, weighing ~300 kg, 104 (male) and 1153 (female) were derived by cloning from a line of primary bovine fibroblasts that had been derived from an Angus–Simmental cross and transfected with a human artificial chromosome that bore the complete unrearranged human lambda (Ig{lambda}) and heavy-chain (IgH) loci. Following a pre-bleed, each calf was injected i.v. with 6 g (20 mg per BWkg) of human IgG (Panglobulin intended for intravenous use, Blood Transfusion Service, Swiss Red Cross, Berne, Switzerland) in 60 ml of saline and during the next 42 days, periodic blood samples were collected in EDTA tubes and evaluated for their content of human IgG. A quantitative ELISA employing an unlabeled affinity-purified bovine anti-human polyclonal antibody as a capture reagent and a biotinylated affinity-purified bovine anti-human polyclonal antibody as a detecting reagent was used to evaluate plasma concentrations of human IgG during the course of the experiment. Samples were assayed in triplicate. The mean human IgG concentration data of the two animals were fitted with a two-compartmental model using WinNonLin Professional, version 4.1 (Pharsight). The individual elimination curves were used to obtain the pharmacokinetic parameters. The individual values were averaged.

All experimental procedures were approved by the Animal 325 Care and Ethics Committee of Hematech, LLC (limited liability company) where all of the animal experiments were conducted using procedures that were deemed in full compliance with the rules of the institutional IACUC (Institutional Animal Care and Use Committee) which was chartered by the federal government of the United States.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
SPR experiments
Recombinant sbFcRn was generated, purified and then analyzed by Coomassie blue staining which detected two bands at molecular weights of 31 and 12 kDa, representing the sbFcRn and bß2m, respectively (data not shown). The interaction of bovine and human IgG with bFcRn and hFcRn at pH 6.0 was investigated using a SPR assay. bFcRn and hFcRn (33) were covalently immobilized onto a biosensor chip, and a series of injections containing 2 nM–10 µM human or bovine IgG were allowed to reach or nearly reach equilibrium (Fig. 1A and B). These data were fit to a model with two classes of non-interacting binding sites, yielding two equilibrium dissociation constants, KD1 and KD2, along with their fraction occupancies, f1 and f2 (Table 1). For the high-affinity population (KD1), which generally accounted for the majority of the binding response, the dissociation constants were 0.45 nM for human IgG over bFcRn, 2.0 nM for bovine IgG over bFcRn, 4.9 nM for human IgG over hFcRn and 430 nM for bovine IgG over hFcRn (Fig. 1 and Table 1).


Figure 1
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 1. Biosensor analyses of bFcRn and hFcRn interacting with bovine and human IgG. (A) Equilibrium binding of bovine and human IgG to bFcRn (1800 RU) immobilized on a biosensor chip is plotted as a function of the log of the IgG concentration. Best-fit binding curves, modeled as two classes of non-interacting binding sites, are superimposed on the binding data. (B) Equilibrium binding of bovine and human IgG to hFcRn (1700 RU).

 

View this table:
[in this window]
[in a new window]
  		Table 1. Binding affinities derived from the data in panels A and B of Fig. 1

 
Generating a stable transfectant bovine cell line (B4) and testing its binding ability to bovine and human IgG
G418-selected bFcRn-transfected cell clones were assessed by western blot using rabbit anti-peptide antisera raised against an epitope of the bFcRn heavy chain (amino acids 173–186) (30). From among several resistant bFcRn/MAC-T clones, we selected one (B4) which strongly expressed a ~40-kDa band which is consistent with the known molecular weight of the bFcRn {alpha} chain (29, 30). There was no hybridization in the untransfected MAC-T cells (Fig. 2A).


Figure 2
View larger version (41K):
[in this window]
[in a new window]
  		Fig. 2. In vitro radioreceptor assays of bFcRn interacting with bovine IgG, human IgG and chicken IgY. (A) Functional expression of bFcRn in transfected (B4) and untransfected (MAC-T) bovine mammary epithelial cell lines. Western blot of total cellular protein (10 µg per lane) by using affinity-purified rabbit antisera raised against amino acids 173–187 of the {alpha}2–{alpha}3 domains (30). (B) Bovine-125I-IgG binding by bFcRn transfected (B4) and untransfected MAC-T cells. Assays were done at 37°C without (filled columns) and with (open columns) competing-unlabeled bovine IgG, at pH 6.0 or 7.5. Each column represents the mean cell-associated radioactivity in three replicates; bars show the standard error of the mean. (C) Binding ability for bovine IgG, human IgG and chicken IgY to transfected MAC-T cells (B4). B4 cells bound bovine and human IgG specifically at pH 6.0 but not at pH 7.5. Human IgG binding was much greater as compared with the bovine IgG uptake. The binding of bovine IgG was completely displaced by non-labeled bovine and human IgG, but not with chicken IgY, added in 1000-fold molar excess. Radiolabeled human IgG was completely displaced by excess of non-labeled human IgGs, but only partially by adding 1000-fold molar excess of non-labeled bovine IgGs.

 
To determine whether the bFcRn/MAC-T clone (B4) expressed a functional FcR, we first measured its binding to radiolabeled bovine IgG. We could detect specific binding at pH 6 in the case of B4 cells, but not in the untransfected control. There was no specific binding at pH 7.5 (Fig. 2B). Next, we analyzed their binding to bovine IgG, human IgG and chicken IgY. We found that B4 cells bound bovine and human IgG specifically at pH 6.0 but not at pH 7.5, and there was no binding of the chicken IgY. Although, the amount and the specific activity of the labeled bovine and human IgGs were similar, human IgG binding was much greater (>10 times) than bovine IgG uptake. The binding of bovine IgG was completely displaced by non-labeled bovine and human IgG, but not by chicken IgY, added in 1000-fold molar excess. This single-point competitive binding assay also revealed that radiolabeled human IgG was completely displaced by excess of non-labeled human IgGs, but only partially by adding 1000-fold molar excess of non-labeled bovine IgGs (Fig. 2C). We have also checked viability of cells before and after binding and found no differences (viability exceeded 98% in both cases).

Although, the single-point competitive binding assay suggested that bFcRn preferably binds human IgG, we performed a more comprehensive analysis to accurately evaluate the binding affinity of these two IgG molecules to the bFcRn. In this assay, the binding of a single concentration of radiolabeled human IgG in the presence of various concentrations of unlabeled human and bovine IgGs was measured by bFcRn-transfected (B4) cells. Strong human and weaker bovine IgG binding was observed, since unlabeled human IgG effectively displaced its radiolabeled form even by ~30-fold molar excess, while bovine IgG did not completely displace the radiolabeled human IgG even at a 1000-fold molar excess (Fig. 3A). Comparison of the IC50 values indicated that human IgG showed an increased in binding to bFcRn at pH 6 of ~43-fold better than the bovine IgG, whereas the IC50 values were 1.52 x 10–9 and 6.6 x 10–8 M for the human and bovine IgG, respectively (Fig. 3B).


Figure 3
View larger version (30K):
[in this window]
[in a new window]
  		Fig. 3. Competitive binding assay of bFcRn interaction with bovine and human IgG by bFcRn-transfected (B4) cells. (A) Human and bovine IgGs were tested in a competitive binding assay to analyze their binding affinity to bFcRn. The assay was performed at pH 6, with increasing amount (2-fold serial dilutions from 1000- to 0.06-fold molar excess) of unlabeled bovine and human IgG, as competitors. (B) The mean radioactivity was plotted against the competitor concentration (M) for each Ig. The data are representative of three independent experiments. The top of the curve is the plateau at a value equal to radiolabeled human IgG binding in the absence of the competing-unlabeled human or bovine IgGs. The bottom of the curve is a plateau equal to non-specific binding. The concentration of unlabeled IgG that produces radioligand binding half-way between the upper and lower plateaus is called the IC50. IC50 values were calculated using Prism for Windows, version 4.0 (GraphPad Software).

 
FcRn detection in an endothelial cell line (bAEC), in tissue capillary endothelial cells and in the kidney
First, reverse transcription–PCR was used to detect bFcRn heavy-chain transcripts from primary bovine arterial endothelial cells that resulted a bFcRn-specific fragment (data not shown). We then analyzed small intestinal tissue and skin sections to reveal their bFcRn expression. FcRn expression was detected in the endothelial cells of the intestinal (duodenum) sub-mucosal connective tissue and skin using affinity-purified anti-FcRn rabbit sera (Fig. 4A and B).


Figure 4
View larger version (77K):
[in this window]
[in a new window]
  		Fig. 4. Immunohistochemical analysis of the cattle small intestine (duodenum), skin and kidney was performed using affinity-purified anti-FcRn rabbit sera. FcRn expression was detected in the endothelial cells of the sub-mucosal connective tissue in the duodenum (A) and subcutaneous connective tissue (B); asterisks indicate blood vessels). In a normal bovine kidney tissue (g, glomerulus; pt, proximal tubule; dt, distal tubule) the assay demonstrated weak, non-specific staining in the glomeruli and granular staining was observed on proximal tubular cells, associated with the basal side. No staining was detected in the interstitium, medulla or distal tubular cells (C). Sections were counterstained using Mayer's hematoxylin. Bars = 50 µm.

 
We subsequently investigated whether, under physiologic conditions, FcRn was expressed in adult kidneys. Normal bovine kidney tissues were stained with an antiserum to bFcRn using immunohistochemistry, and demonstrated weak, non-specific staining in the glomeruli and granular staining on proximal tubular cells, associated with the basal side. No staining was detected in the interstitium, medulla or distal tubular cells (Fig. 4C).

Pharmacokinetics of human IgG and chicken IgY in normal calves
The pharmacokinetic behavior of the human IgG was first examined in four normal calves. Ten milligrams per BWkg human IgG were injected and its levels in serum were measured with a sandwich ELISA. The ELISA was human IgG specific and did not show cross reactivity with bovine Igs. The clearance curves was triphasic, with phase 1 (alpha phase) representing equilibration between the intravascular and extravascular compartments, phase 2 (beta phase) representing a slow elimination, while phase 3 showed an increased rate of removal of human IgG from the intravascular space. Mathematical modeling of phases 1 and 2 until days 6.8 have shown good correlation to the general scheme of FcRn-mediated IgG pharmacokinetics (6), hence we calculated the alpha- and beta-phase half-lives of human IgG in this time frame. The estimated alpha-phase half-lives were 0.22 ± 0.21 (mean ± SD), while the beta-phase half-life was 32.8 ± 1.86 days, based on the two-compartmental modeling analysis. However, the latter data showed a high uncertainty due to the short period of time that could be analyzed (Fig. 5A and Table 2).


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5. Pharmacokinetic parameters. (A) Pharmacokinetic behavior of the human IgG was first examined in normal Holstein calves. A total of 10 mg per BWkg human IgG was injected in the animals and their sera levels were measured with a sandwich ELISA during the course of the experiment. Samples were assayed in triplicate and error bars represent the standard deviation. Black line indicates the first 6.8 days of the mean human IgG concentration of a representative animal which were fitted with a two-compartmental model using WinNonLin (Pharsight). This period is further emphasized by a gray rectangle. Based on this analysis, the estimated human IgG half-life of alpha phase was 0.35 day, while the beta-phase half-life was 32.9 days, although it has a high uncertainty due to the short period that could be analyzed. In the following days, the mean human IgG data were plotted without analysis. Calves generated immune response to the human IgG as it can be seen by the dotted line and open circles. (B) Pharmacokinetics of human IgG in transchromosomic calves. The modeled data (simulated based on the geometric mean of the primary pharmacokinetic parameters) as well as the observed mean serum antibody concentration (µg ml–1) for the two animals were plotted as a function of time (days after injection) of human IgG. Samples were assayed in triplicate and error bars represent the standard deviation. Based on the two-compartmental modeling analysis, the half-life of alpha phase was 0.32 day, while the beta-phase half-life was 33.25 days.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Pharmacokinetic parameters of human IgG in normal (WT-hIgG) and transchromosomic calves (TC-hIgG) as well as chicken IgY in normal calves (WT-cIgY)

 
We also analyzed the anti-human IgG antibody level in the sera of the calves with an ELISA which detected bovine IgG specific to human IgG. We found that anti-human IgG antibody first appeared ~7 days after intravenous administration of human IgG and then its level continuously increased. Following the appearance of endogenous anti-human IgG antibodies, human IgG concentrations in plasma rapidly decreased and therefore presence of these antibodies coincided with phase 3, representing an increased clearance of the human IgG (Fig. 5A).

The pharmacokinetic behavior of the chicken IgY was also analyzed in two normal calves. Chicken IgY level was measured in sera and found a marked acceleration of clearance compared with the human IgG. Due to the fast catabolic rate, alpha and beta phases were not distinguisable. Furthermore, from day 8, there was a sudden drop of the IgY level probably due to antibody response against it. Levels of non-neutralizing antibodies were not assessed in this experiment. The half-life for chicken IgY in these animals was only 2.39 days, analyzed between days 2 (from the presumed steady-state situation after injection) and 6 (Table 2).

Pharmacokinetics of human IgG in transgenic calves
We substantially performed a similar experiment in cloned transchromosomic calves producing human Igs (4), where immunogenicity of human IgG does not pose a problem. Additionally, these animals had relatively low level of endogenous human Igs (human Ig levels prior to injection of human IgG was ~13 µg ml–1) and injecting 20 mg per BWkg human IgG i.v. resulted a concentration of ~200 µg ml–1. We could therefore effectively use this model to accurately analyze the human IgG catabolism in cattle.

The human IgG concentration in these animals was ~200 µg ml–1 after phase 1 of the clearance curve (alpha phase) which correlated well with the human IgG concentration in our first calves in the same phase (~90 µg ml–1), since the latter animals received twice as much human IgG per BWkg. As expected, we did not observe a change in the kinetics of phase 2 (beta phase) due to the onset of the immune response and hence we could analyze the half-life of human IgG in cattle for 42 days. Based on the two-compartmental modeling analysis the half-life of phase 1 was 0.32 day, while the beta-phase half-life was 33.25 days (Fig. 5B and Table 2).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the current study, we have analyzed the role of the bFcRn in IgG catabolism in normal and transchromosomic cattle that produce human Igs (4). We first analyzed the binding affinity of the bovine and human IgGs to the bFcRn on SPR and found that in both cases specific interaction occurred at pH 6, however there was no interaction at pH 7.5. Surprisingly, human IgG bound much stronger to bFcRn than bovine IgG did (Fig. 1 and Table 1). In a control experiment, we found very weak, basically insignificant binding of bovine IgG to hFcRn, reaffirming the previous data (40). In our SPR experiments, FcRn was immobilized rather than IgG to mimic the physiological situation in which membrane-bound FcRn interacts with soluble IgG and to facilitate comparisons with previous biosensor-based assays. The data do not fit well to a one-site model, and were therefore fit to a two-site model, as described in our previous publication (33).

Consistent with the results of the SPR studies, we detected specific binding of both the bovine and human IgG to the bFcRn stably transfected epithelial cells at pH 6, but not at pH 7.5, in a single-point competitive binding assay (Fig. 2). Noteworthy, the increased binding of human IgG to bFcRn at pH 6 did not exhibit a parallel increase in binding at pH 7.5 (Figs 1 and 2), hence human IgG should not be trapped in cells that express FcRn, as occurs with some mutant human IgG1 molecules in mice (41). We could not observe specific interaction between chicken IgY and bFcRn. It has been long known, that mammalian FcRn does not bind to IgY (42, 43) and mammalian IgGs do not transfer into the chick embryo (44), suggesting that a distinct receptor might be involved in IgY transport. This receptor, a phospholipase A2R homolog, has been recently isolated and characterized (45).

In a competitive binding assay, comparison of the IC50 values indicated that human IgG showed an increase in binding to bFcRn at pH 6 of ~40-fold better than the bovine IgG (Fig. 3). This result correlates well with our SPR results (Fig. 1 and Table 1), which indicated that the affinity of bFcRn for human IgG (KD1 = 0.45 nM) was many times higher than for bovine IgG (KD1 = 2 nM). The difference in binding affinities between bovine and human IgG to bFcRn, analyzed by the in vitro receptor assay is greater than that we found by, possibly due to the condition we used. FcRn is localized primarily intracellularly in epithelial and endothelial cells and only a small fraction of it participates in IgG binding on the cell surface (43, 46). It is also directly involved in exocytic events involving transported IgG at the plasma membrane (47). In our case, the receptor assays occurred at pH 6 which allowed more efficient IgG binding to the surface bFcRn in the transfected MAC-T cells and hence, more effective uptake relative to the untransfected MAC-T cells where only fluid phase processes (Fig. 2B). Furthermore, the condition of the assay (pH 6) results poor release of the IgG during exocytic events involving FcRn at the plasma membrane. Thus, the IgG that has higher affinity to the receptor accumulates at substantially higher level in the cells, as we observed in the case of human IgG comparing it to the bovine IgG (Fig. 2C). The situation described here reflects a recent finding, in which the binding characteristics of a mutated human IgG that has higher affinity and reduced pH dependence to FcRn were analyzed (48). Although, the binding experiments in that case were performed at pH 7.2, the reduced pH dependence of the mutated IgG allowed effective binding to the cell surface and poor release during exocytosis, resulting in similarly high accumulation of the mutated molecule (48). As our competitive binding assay was also performed at pH 6, the competition between human and bovine IgGs occurred primarily at the cell surface due to the FcRn molecules that locate originally, albeit at low concentrations, in the membrane and to those receptors that are involved in the exocytosis and thus the data we got reflects to the affinity difference between human and bovine IgGs (Fig. 3).

Biochemical studies of membrane-bound and soluble forms of FcRn demonstrate that IgG binding is pH dependent: there is strong binding (nanomolar KD) under the acidic conditions (pH ~6.0) found in endosomal compartments and in rodent intestines, while at the slightly basic pH of blood (pH 7.4), there is no detectable binding (14). From mutagenesis and crystallographic studies, the mechanism of this pH dependence has been shown to involve chemical rather than conformational changes (49–51). Specifically, there are attractive interactions at acidic pH between protonated histidines on Fc and negatively charged side chains on FcRn, which are lost upon deprotonation of the Fc histidines at basic pH (50). The sequences of FcRn genes isolated from other organisms, including pig (52), sheep (30), macaque (GenBank accession number AAL92101), brushtail possum (53) and cow (29), suggest that the features described above for rodent and hFcRn proteins are shared in the other mammalian orthologs. A comparison between rat, mouse, hFcRn and bFcRn alpha chain and ß2m residues which are supposed to be involved in binding to IgG molecules have been extensively analyzed based on a crystallography analysis of a rat FcRn-heterodimeric Fc complex (50). This study compared important residues in the interaction and found that amino acid residues of rat Glu117 and Glu132 are conserved (although Glu132 is replaced by Asp132 in humans and cows), indicating that the two pH-dependent salt bridges involving amino acid residues His310 and His435 of the IgG/Fc would still form at pH 6. Asp137 is not conserved, as the hFcRn sequence has Leu (neutral) and the bFcRn has Arg at this position. In the rat, the FcRn Asp137 interacts with His436 of IgG, however, His436 is not conserved in all Fc{gamma} chains and neither the human nor the bovine has His at that position (Tyr or Phe in human Fc{gamma} chains and Tyr in the bovine Fc{gamma} chains). It is interesting that the counterpart of Asp137 is a positively charged residue (Arg) in the cattle [and also in the sheep and pig sequences (30, 52)], which would imply that it cannot be interacting with a positively charged His on IgG Fc, so the prediction would be that bFcRn should not bind any rat IgG subclass as they all have His at position 436. Another residue Trp133, which is a solvent-exposed tryptophan in FcRn that is required for binding between rat FcRn and Fc, is conserved in all sequences analyzed so far. Residues 250 and 251, both are histidines in rat FcRn and interact with a negatively charged residue, Glu89, on rat ß2m in the FcRn dimer, which have been detected in some crystal forms (54). Positions 250 and 251 are conserved as His in all FcRn sequences, however, ß2m position 89 is not conserved as human and bovine have Gln at this position, and hence this interaction does not take place in all species. Based on the similarities between hFcRn and bFcRn heavy chain and ß2m sequences, listed above, one would not predict an increased affinity of the bFcRn to human IgG.

Earlier pharmacokinetic studies have indicated that the catabolic site for IgG and other proteins was most likely the vascular endothelium (5). Distribution studies of murine IgG1, Fc fragments and anti-FcRn antibodies have demonstrated that the major sites of FcRn function in adult, non-pregnant mice are skin and muscle, with a lesser involvement of liver and adipose tissue (20). Supportive of this hypothesis are the findings that FcRn alpha chain is expressed in many tissues, and it has been detected in both mouse and human endothelial cells (20, 55). In accordance with these results, we could amplify FcRn alpha-chain cDNA from primary bAEC and also show its expression in capillary endothelial cells of the skin and small intestine (Fig. 4). FcRn is also expressed in human renal glomerular epithelial cells and in the brush border of the proximal tubular cells (56). More recently, it has been demonstrated that monolayers of a cell line derived from proximal tubular cells are able to transcytose intact IgG (57). These data led to the suggestion that FcRn may play a role in the reabsorption of filtered IgG, and hence the minimization of the role of urinary excretion as a route of IgG elimination (6). We demonstrated the expression of FcRn alpha-chain mRNA in a bovine kidney epithelial cell line (MDBK) in our earlier study (29). Here we have shown the presence of the FcRn alpha chain in the basal side of the proximal tubular cells of the kidney, but not in the glomerular epithelial cells (Fig. 4). Although, the cellular localization of bFcRn is different in the proximal tubular epithelial cells compared with its human counterpart (56), we suggest that the function should be similar, as urine in healthy cattle also contains only trace amounts of IgG (28).

The elimination of IgG is known to be concentration dependent, where half-life decreases as a function of increasing serum IgG concentrations (5). Therefore, we used a single intravenous injection of human IgG dose of 10 and 20 mg per BWkg in our in vivo models, since this amount is considered to lead to insignificant changes in the apparent elimination rate constant of IgG (6). As with other drugs, the overall extent of distribution of the injected Ig is typically quantified by inferring the ratio of the mass of the Ig in the body at the steady state and the concentration of Ig in plasma at steady state (58). For most antibodies, tissue : blood concentration ratios are found to be in the range of 0.1–0.5 (59, 60). Given that the plasma volume is 3–5% of the total body volume, Ig in plasma are expected to comprise ~20–50% of all antibodies in the body (6). In our study, the plasma steady-state concentration (2–3 days after injection) of the human IgG was ~90 µg ml–1 and ~200 µg ml–1 in normal and transchromosomic calves, respectively (Fig. 5). These values indicate that the plasma of these calves contained 30–50% of the injected human IgG, which fits well to the model in which antibody does not bind with high affinity to extravascular sites (6) and also to an earlier study suggesting that bovine IgG1 and IgG2 were divided almost equally between the intra- and extravascular pools before parturition, in cows (61).

In normal calves, the injected human IgG led to an immune response which we could detect at ~7 days following administration, with a peak antibody concentration observed at ~16 days. We also found that the presence of anti-human IgG antibodies led to an increased elimination rate of the human IgG (Fig. 5). These results are in good agreement with previous studies, regarding the time course (62) and pharmacokinetic effect of induced antibody responses (63). Determination of the human IgG beta-phase half-life resulted in a value of ~33 days (Fig. 5A and Table 2), although with high uncertainty, due to the relatively short period. We also found that the chicken IgY, which does not bind to bFcRn (Fig. 2), is cleared rapidly (Table 2), as it has been indicated in previous studies in cattle (64) and mouse (17).

To accurately analyze the catabolism of human IgG in cattle, we used transchromosomic calves, in which human IgG administration does not elicit an antibody response. Therefore, we did not observe a change in the kinetics of the beta phase and hence, we could accurately analyze the mean elimination (beta phase) half-life of human IgG in a relatively long time scale, in these animals. Based on these results, the beta-phase half-life of the human IgG was ~33 days (Fig. 5B and Table 2), which is 1.5–3 times longer compared with its bovine counterpart, supposing that the beta-phase half-life of bovine IgG is in the region of 10–22 days (24, 25). This result agrees well with data of a most recent study in which mutated human IgG2 molecules, which bound 28-fold stronger to hFcRn as compared with wild-type antibody at pH 6, but did not interact at pH 7.5, showed a 2-fold longer serum half-life in rhesus monkeys (19). Worth mentioning, that human IgG binds much stronger to the bFcRn as compared with the hFcRn (>10 order magnitude, Fig. 1 and Table 1) which is also in good agreement to the longer half-life of human IgG in cattle in contrast to its half-life in humans (21 days).

In summary, we have demonstrated that bFcRn binds both bovine and human IgGs in a pH-dependent manner. Furthermore, bFcRn is expressed, among other tissues, in capillary endothelial cells and in the kidney which are involved in IgG metabolism. We could also show a longer half-life for the human IgG in calves, indicating that there is correlation between pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives, as has been previously demonstrated in mice and rhesus monkeys. These data suggest that FcRn is involved in IgG homeostasis in cattle and furthermore, that transchromosomic calves producing human Igs can effectively protect their human IgG. The latter has implications for the successful large-scale production of therapeutic antibodies in these animals. Future studies should be aimed at testing the different human and bovine IgG isotypes interaction with the bFcRn, the role of bFcRn in secreting IgG onto the mucosal surfaces and into colostrum/milk and how this process influences human or bovine IgG metabolism in these animals.


   Acknowledgements
 
We thank Viktória Juhász for exellent technical assistance in the competitive binding assays on stably transfected cells. We also thank for Drs. Csaba Bajcsy and András Horváth for their help with the normal calf experiments. This study was supported by grants from the Ministry of Education, Republic of Hungary Grant (OMFB 01605/2002) and National Research Fundation of Hungary (OTKA 049015) to I.K., National Institutes of Health (R37 AI041239-07) to P.J.B. and NSF-MCB 0131335 to R.A.G.


   Abbreviations
 
bAEC   bovine aortic endothelial cell
bFcRn   bovine neonatal Fc receptor
bß2m   bovine beta 2-microglobulin
BWkg   body weight kilogram
ß2m   beta 2-microglobulin
FcRn   neonatal Fc receptor
hFcRn   human neonatal Fc receptor
IC50   inhibitory concentration 50%
i.v.   intravenously
RU   resonance unit
sbFcRn   soluble bovine FcRn
SPR   surface plasmon resonance

   Notes
 
Transmitting editor: A. Falus

Received 19 October 2005, accepted 3 January 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Bruggemann, M. and Neuberger, M. S. 1996. Strategies for expressing human antibody repertoires in transgenic mice. Immunol. Today 17:391.[CrossRef][ISI][Medline]
  2. Tomizuka, K., Shinohara, T., Yoshida, H. et al. 2000. Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc. Natl Acad. Sci. USA 97:722.[Abstract/Free Full Text]
  3. Magadan, S., Valladares, M., Suarez, E. et al. 2002. Production of antigen-specific human monoclonal antibodies: comparison of mice carrying IgH/kappa or IgH/kappa/lambda transloci. Biotechniques 33:680.
  4. Kuroiwa, Y., Kasinathan, P., Choi, Y. J. et al. 2002. Cloned transchromosomic calves producing human immunoglobulin. Nat. Biotechnol. 20:889.[CrossRef][ISI][Medline]
  5. Waldmann, T. A. and Strober, W. 1969. Metabolism of immunoglobulins. Prog. Allergy 13:1.[ISI][Medline]
  6. Lobo, E. D., Hansen, R. J. and Balthasar, J. P. 2004. Antibody pharmacokinetics and pharmacodynamics. J. Pharm. Sci. 93:2645.[CrossRef][ISI][Medline]
  7. Ghetie, V. and Ward, E. S. 2002. Transcytosis and catabolism of antibody. Immunol. Res. 25:97.[CrossRef][ISI][Medline]
  8. Kuus-Reichel, K., Grauer, L. S., Karavodin, L. M., Knott, C., Krusemeier, M. and Kay, N. E. 1994. Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies? Clin. Diagn. Lab. Immunol. 1:365.
  9. Wawrzynczak, E. J., Cumber, A. J., Parnell, G. D., Jones, P. T. and Winter, G. 1992. Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the CH2 domains. Mol. Immunol. 29:213.[CrossRef][Medline]
  10. Meier, W., Gill, A., Rogge, M. et al. 1995. Immunomodulation by LFA3TIP, an LFA-3/IgG1 fusion protein: cell line dependent glycosylation effects on pharmacokinetics and pharmacodynamic markers. Ther. Immunol. 2:159.[Medline]
  11. Newkirk, M. M., Novick, J., Stevenson, M. M., Fournier, M. J. and Apostolakos, P. 1996. Differential clearance of glycoforms of IgG in normal and autoimmune-prone mice. Clin. Exp. Immunol. 106:259.[Medline]
  12. Gillies, S. D., Lo, K. M., Burger, C., Lan, Y., Dahl, T. and Wong, W. K. 2002. Improved circulating half-life and efficacy of an antibody-interleukin 2 immunocytokine based on reduced intracellular proteolysis. Clin. Cancer Res. 8:210.[Abstract/Free Full Text]
  13. Simister, N. E. and Mostov, K. E. 1989. An Fc receptor structurally related to MHC class I antigens. Nature 337:184.[CrossRef][Medline]
  14. Ghetie, V. and Ward, E. S. 2000. Multiple roles for the major histocompatibility complex class I- related receptor FcRn. Annu. Rev. Immunol. 18:739.[CrossRef][ISI][Medline]
  15. Ghetie, V., Hubbard, J. G., Kim, J. K., Tsen, M. F., Lee, Y. and Ward, E. S. 1996. Abnormally short serum half-lives of IgG in beta 2-microglobulin-deficient mice. Eur. J. Immunol. 26:690.[ISI][Medline]
  16. Junghans, R. P. and Anderson, C. L. 1996. The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor. Proc. Natl Acad. Sci. USA 93:5512.[Abstract/Free Full Text]
  17. Israel, E. J., Wilsker, D. F., Hayes, K. C., Schoenfeld, D. and Simister, N. E. 1996. Increased clearance of IgG in mice that lack beta 2-microglobulin: possible protective role of FcRn. Immunology 89:573.[CrossRef][ISI][Medline]
  18. Ghetie, V., Popov, S., Borvak, J. et al. 1997. Increasing the serum persistence of an IgG fragment by random mutagenesis. Nat. Biotechnol. 15:637.[CrossRef][ISI][Medline]
  19. Hinton, P. R., Johlfs, M. G., Xiong, J. M. et al. 2004. Engineered human IgG antibodies with longer serum half-lives in primates. J. Biol. Chem. 279:6213.[Abstract/Free Full Text]
  20. Borvak, J., Richardson, J., Medesan, C. et al. 1998. Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice. Int. Immunol. 10:1289.[Abstract/Free Full Text]
  21. Chaudhury, C., Mehnaz, S., Robinson, J. M. et al. 2003. The major histocompatibility complex–related Fc receptor for IgG (FcRn) binds albumin and prolongs its lifespan. J. Exp. Med. 197:315.[Abstract/Free Full Text]
  22. Kim, J., Bronson, C. L., Hayton, W. L. et al. 2005. Albumin turnover: FcRn-mediated recycling saves as much albumin from degradation as the liver produces. Am. J. Physiol. Gastrointest. Liver Physiol 290:G352.
  23. Butler, J. E. 1986. Biochemistry and biology of ruminant immunoglobulins. Prog. Vet. Microbiol. Immunol. 2:1.[Medline]
  24. Brandon, M. R. and Lascelles, A. K. 1971. Relative efficiency of absorption of IgG1, IgG2, IgA and IgM in the newborn calf. Aust. J. Exp. Biol. Med. Sci. 49:629.[Medline]
  25. Nielsen, K., Sheppard, J., Holmes, W. and Tizard, I. 1978. Experimental bovine trypanosomiasis. Changes in the catabolism of serum immunoglobulins and complement components in infected cattle. Immunology 35:811.[Medline]
  26. Nansen, P. 1970. Metabolism of bovine immunoglobulin-G. Munksgaard, Copenhagen.
  27. Husband, A. J., Brandon, M. R. and Lascelles, A. K. 1972. Absorption and endogenous production of immunoglobulins in calves. Aust. J. Exp. Biol. Med. Sci. 50:491.[Medline]
  28. Butler, J. E. 1983. Bovine immunoglobulins: an augmented review. Vet. Immunol. Immunopathol. 4:43.[CrossRef][Medline]
  29. Kacskovics, I., Wu, Z., Simister, N. E., Frenyo, L. V. and Hammarstrom, L. 2000. Cloning and characterization of the bovine MHC class I-like Fc receptor. J. Immunol. 164:1889.[Abstract/Free Full Text]
  30. Mayer, B., Zolnai, A., Frenyo, L. V. et al. 2002. Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs. Immunology 107:288.[CrossRef][Medline]
  31. Mayer, B., Kis, Z., Kajan, G., Frenyo, L. V., Hammarstrom, L. and Kacskovics, I. 2004. The neonatal Fc receptor (FcRn) is expressed in the bovine lung. Vet. Immunol. Immunopathol. 98:85.[Medline]
  32. Ellis, S. A., Braem, K. A. and Payne, L. K. 1993. Nucleotide sequence of cattle beta 2-microglobulin cDNA. Immunogenetics 38:310.[Medline]
  33. West, A. P., Jr and Bjorkman, P. J. 2000. Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor. Biochemistry 39:9698.[CrossRef][Medline]
  34. Vaughn, D. E. and Bjorkman, P. J. 1997. High-affinity binding of the neonatal Fc receptor to its IgG ligand requires receptor immobilization. Biochemistry 36:9374.[CrossRef][Medline]
  35. Mackenzie, N. 1984. Fc receptor-mediated transport of immunoglobulin across the intestinal epithelium of the neonatal rodent. Immunol. Today 5:364.[CrossRef]
  36. Israel, E. J., Simister, N., Freiberg, E., Caplan, A. and Walker, W. A. 1993. Immunoglobulin G binding sites on the human foetal intestine: a possible mechanism for the passive transfer of immunity from mother to infant. Immunology 79:77.[Medline]
  37. Ausubel, F. M., Brent, R., Kingston, R. E. et al. 1989. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons., Hoboken, NJ, USA.
  38. Gorman, C. M., Gies, D. R. and McCray, G. 1990. Transient production of proteins using an adenovirus transformed cell line. DNA Protein Eng. Tech. 2:3.
  39. Story, C. M., Mikulska, J. E. and Simister, N. E. 1994. A major histocompatibility complex class I-like Fc receptor cloned from human placenta: possible role in transfer of immunoglobulin G from mother to fetus. J. Exp. Med. 180:2377.[Abstract/Free Full Text]
  40. Ober, R. J., Radu, C. G., Ghetie, V. and Ward, E. S. 2001. Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies. Int. Immunol. 13:1551.[Abstract/Free Full Text]
  41. Dall' Acqua, W. F., Woods, R. M., Ward, E. S. et al. 2002. Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences. J. Immunol. 169:5171.[Abstract/Free Full Text]
  42. Halliday, R. 1955. The absorption of antibodies from immune sera by the gut of the young rat. Proc. R. Soc. Lond. B 143:408.[Medline]
  43. McCarthy, K. M., Yoong, Y. and Simister, N. E. 2000. Bidirectional transcytosis of IgG by the rat neonatal Fc receptor expressed in a rat kidney cell line: a system to study protein transport across epithelia. J. Cell Sci. 113:1277.[Abstract]
  44. Brambell, F. W. R. 1970. The Transmission of Passive Immunity from Mother to Young. North-Holland Publishing, Amsterdam.
  45. West, A. P., Jr, Herr, A. B. and Bjorkman, P. J. 2004. The chicken yolk sac IgY receptor, a functional equivalent of the mammalian MHC-related Fc receptor, is a phospholipase A(2) receptor homolog. Immunity 20:601.[CrossRef][ISI][Medline]
  46. Ober, R. J., Martinez, C., Vaccaro, C., Zhou, J. and Ward, E. S. 2004. Visualizing the site and dynamics of IgG salvage by the MHC class I-related receptor, FcRn. J. Immunol. 172:2021.[Abstract/Free Full Text]
  47. Ober, R. J., Martinez, C., Lai, X., Zhou, J. and Ward, E. S. 2004. Exocytosis of IgG as mediated by the receptor, FcRn: an analysis at the single-molecule level. Proc. Natl Acad. Sci. USA 101:11076.[Abstract/Free Full Text]
  48. Vaccaro, C., Zhou, J., Ober, R. J. and Ward, E. S. 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat. Biotechnol. 23:1283.[CrossRef][ISI][Medline]
  49. Kim, J. K., Tsen, M. F., Ghetie, V. and Ward, E. S. 1994. Localization of the site of the murine IgG1 molecule that is involved in binding to the murine intestinal Fc receptor. Eur. J. Immunol. 24:2429.[ISI][Medline]
  50. Martin, W. L., West, A. P., Jr, Gan, L. and Bjorkman, P. J. 2001. Crystal structure at 2.8 A of an FcRn/heterodimeric Fc complex: mechanism of pH-dependent binding. Mol. Cell 7:867.[CrossRef][ISI][Medline]
  51. Vaughn, D. E. and Bjorkman, P. J. 1998. Structural basis of pH-dependent antibody binding by the neonatal Fc receptor. Structure 6:63.[Medline]
  52. Schnulle, P. M. and Hurley, W. L. 2003. Sequence and expression of the FcRn in the porcine mammary gland. Vet. Immunol. Immunopathol. 91:227.[CrossRef][Medline]
  53. Adamski, F. M., King, A. T. and Demmer, J. 2000. Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum). Mol. Immunol. 37:435.[CrossRef][ISI][Medline]
  54. Burmeister, W. P., Gastinel, L. N., Simister, N. E., Blum, M. L. and Bjorkman, P. J. 1994. Crystal structure at 2.2 A resolution of the MHC-related neonatal Fc receptor. Nature 372:336.[CrossRef][Medline]
  55. Ward, E. S., Zhou, J., Ghetie, V. and Ober, R. J. 2003. Evidence to support the cellular mechanism involved in serum IgG homeostasis in humans. Int. Immunol. 15:187.[Abstract/Free Full Text]
  56. Haymann, J. P., Levraud, J. P., Bouet, S. et al. 2000. Characterization and localization of the neonatal Fc receptor in adult human kidney. J. Am. Soc. Nephrol. 11:632.[Abstract/Free Full Text]
  57. Kobayashi, N., Suzuki, Y., Tsuge, T., Okumura, K., Ra, C. and Tomino, Y. 2002. FcRn-mediated transcytosis of immunoglobulin G in human renal proximal tubular epithelial cells. Am. J. Physiol. Renal Physiol. 282:F358.[Abstract/Free Full Text]
  58. Gibaldi, M. and Perrier, D. 1982. Pharmacokinetics, 2nd Rev Ex edn. Marcel Dekker, New York.
  59. Zhu, H., Melder, R. J., Baxter, L. T. and Jain, R. K. 1996. Physiologically based kinetic model of effector cell biodistribution in mammals: implications for adoptive immunotherapy. Cancer Res. 56:3771.[Abstract/Free Full Text]
  60. Molthoff, C. F., Pinedo, H. M., Schluper, H. M., Nijman, H. W. and Boven, E. 1992. Comparison of the pharmacokinetics, biodistribution and dosimetry of monoclonal antibodies OC125, OV-TL 3, and 139H2 as IgG and F(ab')2 fragments in experimental ovarian cancer. Br. J. Cancer 65:677.[Medline]
  61. Sasaki, M., Davis, C. L. and Larson, B. L. 1976. Production and turnover of IgG1 and IgG2 immunoglobulins in the bovine around parturition. J. Dairy Sci. 59:2046.[Abstract/Free Full Text]
  62. Gobburu, J. V., Tenhoor, C., Rogge, M. C. et al. 1998. Pharmacokinetics/dynamics of 5c8, a monoclonal antibody to CD154 (CD40 ligand) suppression of an immune response in monkeys. J. Pharmacol. Exp. Ther. 286:925.[Abstract/Free Full Text]
  63. Richter, W. F., Gallati, H. and Schiller, C. D. 1999. Animal pharmacokinetics of the tumor necrosis factor receptor-immunoglobulin fusion protein lenercept and their extrapolation to humans. Drug Metab. Dispos. 27:21.[Abstract/Free Full Text]
  64. Erhard, M. H., Gobel, E., Lewan, B., Losch, U. and Stangassinger, M. 1997. Systemic availability of bovine immunoglobulin G and chicken immunoglobulin Y after feeding colostrum and whole egg powder to newborn calves. Arch. Tierernahr. 50:369.[Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles: