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Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding 2

Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 7, pp. 4826 –4836, February 12, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in □ S Immunoglobulin G and Albumin Binding Received for publication, November 4, 2009, and in revised form, December 3, 2009 Published, JBC Papers in Press, December 14, 2009, DOI 10.1074/jbc.M109.081828 ‡1 ‡ ‡ §¶ ‡2 Jan Terje Andersen , Muluneh Bekele Daba , Gøril Berntzen , Terje E. Michaelsen , and Inger Sandlie ‡ § From the Department of Molecular Biosciences and Centre for Immune Regulation and Institute of Pharmacy, University of Oslo, P.O. Box 1041, N-0371 Oslo and the Norwegian Institute of Public Health, N-0403 Oslo, Norway The neonatal Fc receptor (FcRn) regulates the serum half-life half-life, transport of IgG across intestinal epithelia and pla- of both IgG and albumin through a pH-dependent mechanism centa, as well as enhancement of neutrophil phagocytosis of that involves salvage from intracellular degradation. Therapeu- immune complexes, as reviewed previously (1). Moreover, the tics and diagnostics built on IgG, Fc, and albumin fusions are receptor plays a role in antibody-mediated antigen presenta- frequently evaluated in rodents regarding biodistribution and tion by dendritic cells (2). FcRn has also been found to salvage pharmacokinetics. Thus, it is important to address cross-species albumin from intracellular degradation (3), in a fashion similar ligand reactivity with FcRn, because in vivo testing of such mol- to that described for IgG, which involves receptor ligand inter- ecules is done in the presence of competing murine ligands, both actions in acidified endosomal compartments (1). Hence, FcRn in wild type (WT) and human FcRn (hFcRn) transgenic mice. affects diverse and important immunological and non-immu- Here, binding studies were performed in vitro using enzyme- nological processes. linked immunosorbent assay and surface plasmon resonance FcRn is a heterodimeric receptor consisting of a transmem- WT with recombinant soluble forms of human (shFcRn ) and brane heavy chain (HC) that is non-covalently associated with WT mouse (smFcRn ) receptors. No binding of albumin from 2-microglobulin (2m). Consequently, the significance of either species was observed at physiological pH to either recep- FcRn has been extensively documented in knockout mouse tor. At acidic pH, a 100-fold difference in binding affinity was models lacking 2m or the HC. Such deficient mice have IgG WT observed. Specifically, smFcRn bound human serum albumin serum levels of 20–30% that of wild-type mice and a 60% WT with a K of 90 M, whereas shFcRn bound mouse serum reduced level of MSA (3, 4). A human example is the rare famil- WT albumin with a K of 0.8 M. shFcRn ignored mouse IgG1, ial hypercatabolic hypoproteinemia syndrome that is charac- WT and smFcRn bound strongly to human IgG1. The latter pair terized by reduced serum levels of both hIgG and HSA (5). An also interacted at physiological pH with calculated affinity in the explanation was provided when deficient FcRn expression was micromolar range. In all cases, binding of albumin and IgG from demonstrated as a result of a point mutation in the 2m-encod- either species to both receptors were additive. Cross-species ing gene sequence that disrupts efficient secretion (6). Thus, albumin binding differences could partly be explained by non- FcRn is truly bifunctional and contributes to maintaining the conserved amino acids found within the 2-domain of the high levels of IgG as well as albumin in serum, with levels receptor. Such distinct cross-species FcRn binding differences amounting to 12 and 40 mg/ml, respectively, in mice and must be taken into consideration when IgG- and albumin-based humans. therapeutics and diagnostics are evaluated in rodents for their The FcRn HC consists of three ectodomains (1, 2, and 3), pharmacokinetics. a short transmembrane region, and a cytoplasmic tail (1). Mutagenesis and crystallographic studies have uncovered that the FcRn-IgG interaction is mediated by Fc-localized residues, The major histocompatibility class I-related neonatal Fc especially Ile-253, His-310, and His-435, and acidic surface- receptor (FcRn) is a versatile receptor that regulates serum IgG exposed residues on the 2-domain of the HC (7–9). The inter- action is strictly pH-dependent with binding at acidic pH and * This work was supported by grants from the Steering Board for Research in no or very weak binding at physiological pH. The histidines are Molecular Biology, Biotechnology and Bioinformatics at the University of mainly responsible for the pH dependence, because they are Oslo (to J. T. A.), The Norwegian Research Council (Grant 179573), and the Norwegian Cancer Society (Grant B95078). protonated under acidic conditions. Although the FcRn-albu- □ S The on-line version of this article (available at http://www.jbc.org) contains min interaction is less well characterized, data indicate that supplemental Figs. 1– 4 and Tables 1–3. domain III of albumin binds to the HC 2-domain at a site To whom correspondence may be addressed. Tel.: 47-22-85-47-93; Fax: 47-22-85-40-61; E-mail: [email protected]. distant from the IgG binding site, because His-166 is crucial for To whom correspondence may be addressed. E-mail: [email protected]. the interaction (10, 11). Thus, both ligands may bind simulta- The abbreviations used are: FcRn, neonatal Fc receptor; HC, heavy chain; HEK, neously in a pH-dependent manner. humanembryonickidney;hFcRn,humanFcRn;hIgG,humanIgG;HSA,human serum albumin; h2m, human 2-microglobulin; mIgG, mouse IgG; MSA, Knowledge of FcRn-IgG biology explains the prolonged half- mouse serum albumin; oriP, origin of replication; RU, resonance unit; shFcRn, life of IgG Fc-fused therapeutics (1, 4, 12, 13). Understanding of soluble hFcRn; smFcRn, soluble mouse FcRn; SPR, surface plasmon resonance; the FcRn-IgG interaction at the atomic level has prompted the WT, wild type; NIP, 3-iodo-4-hydroxy-5-nitrophenacetyl; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase. development of novel IgG-based therapeutics with point muta- 4826 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 7 •FEBRUARY 12, 2010 This is an Open Access article under the CC BY license. Cross-species Binding to FcRn tions in their Fc part that modulate serum half-life (14–19). Construction of Mutant FcRn Variants—A single amino acid- Furthermore, improved half-life and efficiency of a number of substituted mFcRn variant was constructed by mutating His- 168 to alanine by site-directed mutagenesis using the plasmid small therapeutic molecules and proteins that are normally WT pcDNA3-mFcRn -GST-h2m-oriP and the primers cleared rapidly from the circulation have been achieved by strategies such as chemical conjugation or genetic fusion to mFcRnH168AForw and mFcRnH168ARev. Three double mutant E117A/E118A R164L/E165G albumin itself (20–25) or any of several albumin binding mol- FcRn variants, named hFcRn , hFcRn , and L166R/G168E mFcRn , were constructed using the templates ecules (26–29). WT WT pcDNA3-mFcRn -GST-h2m-oriP and pcDNA3-hFcRn - Mice are routinely used as convenient first line models for GST-h2m-oriP. The primer sequences used are all listed in preclinical evaluation of such therapeutics. Thus, it is crucial to supplemental Table 1. understand if and how mFcRn interacts with human ligands. Expression and Purification of Soluble FcRn Variants—For Indeed, mFcRn has been shown to be rather promiscuous in its transient transfections, the hFcRn- and mFcRn-encoding plas- binding to IgG. It binds IgG from different species, including mids were transfected into HEK 293E cells (ATCC) using Lipo- hIgG. On the other hand, hFcRn discriminates binding to mIgG fectamine 2000 (Invitrogen) following the manufacturer’s (except for weak binding to mIgG2b) (30). This latter finding instructions. HEK 293E cells were cultured in Dulbecco’s mod- has greatly contributed to the understanding of the fast clear- ified Eagle’s medium (BioWhittaker) using standard condi- ance and disappointing therapeutic effects obtained using tions. Pooled media were filtrated and applied on a GSTrap FF monoclonal mIgGs in human trials. However, fast or interme- column (5 ml, Amersham Biosciences) connected to a semiau- diate clearance can also be favorable, as demonstrated for IgG tomatic workstation and recorder, and purifications were per- immunoconjugates approved for cancer imaging and therapy formed essentially as recommended in the manufacturer’s (16, 31). manual. Eluted fractions were pooled, concentrated, and ana- Mice have recently been constructed that lack the mFcRn lyzed under non-reducing or reducing condition using -mer- HC and are transgenic for the human counterpart (4). Such captoethanol (Sigma-Aldrich). Samples of 2 g of each receptor mice express hFcRn that is exposed to the murine ligands, were applied on a 12% SDS-PAGE (Bio-Rad). Protein concen- mIgG and MSA. Interestingly, they are found to be unable to trations were determined using a NanoDrop N-1000 spectro- protect mIgG from degradation. Nothing is known about the photometer (NanoDrop Technologies). cross-species interaction between FcRn and albumin. Construction, Production, and Purification of IgG Variants— Herein, we report on important cross-species ligand-FcRn WT A mouse plasmacytoma cell line producing chimeric human binding differences. Specifically, at acidic pH shFcRn binds IgG1 (hIgG1) anti-3-iodo-4-hydroxy-5-nitrophenacetyl (NIP) MSA strongly while ignoring mIgG binding, whereas WT was a gift from Dr. M. Neuberger (Medical Research Council smFcRn binds hIgG1 strongly and HSA very weakly. The Laboratory of Molecular Biology, Cambridge, UK). The con- cross-species differences in albumin binding could partly be struction of this antibody has been described before (33). Pure explained by non-conserved amino acid variations found in the preparations of anti-NIP mIgG1 and mIgG2b were gifts from vicinity of the conserved His-166 of the HC. In vivo, the conse- WT Dr. Gregory Winter (Centre for Protein Engineering, Medical quences of weak binding of HSA to smFcRn may facilitate Research Council Centre, UK). A single amino acid-substituted rapid clearance in the presence of high amounts of endogenous chimeric hIgG1 variant was constructed by mutating His-435 MSA. Such cross-species kinetic differences have great rele- (numbering according to the EU index) to alanine by site-di- vance for preclinical pharmacokinetics and biodistribution rected mutagenesis using the primers hIgG1H435Aforw and evaluations of engineered therapeutic and diagnostic IgGs, Fc, hIgG1H435Arev (listed in supplemental Table 1) and the tem- and albumin fusions in rodents. plate vector pLNOH2/C1 (34), which contains the gene frag- ment encoding the constant HC of hIgG1. The mutant vector EXPERIMENTAL PROCEDURES H435A denoted pLNOH-hIgG1 was transiently expressed in PCR and Subcloning—The cDNA segments encoding trun- HEK 293E cells by co-transfection with the pLNOK vector cated hFcRn HC and human 2m (h2m) were PCR-amplified encoding the mouse lambda light chain as above. Chimeric H435A from a U937 cell line (ATCC) cDNA library followed by sub- hIgG1 was purified on NIP-coupled Sepharose as previ- cloning of the fragments into the pcDNA3-GST vector, all as ously described (35). The integrity of expressed protein was previously described (32). A mouse liver cDNA library (Zyagen) verified by non-reducing SDS-PAGE analyses followed by was used to PCR-amplify a cDNA encoding a truncated version Western blotting using a horseradish peroxidase-conjugated of the mFcRn HC (encoding the endogenous native leader polyclonal rabbit anti-human Fc (Amersham Biosciences) and sequence, 1, 2, and 3 domains; 293 amino acids) using the horseradish peroxidase-conjugated anti-murine lambda light primers mFcRnForw and mFcRnRev, listed in supplemental chain (Southern Biotech) (data not shown). Table 1. Primers were designed to allow in-frame ligation of the Size-exclusion Chromatography Purification of Albumin fragment upstream of a cDNA encoding a glutathione S-trans- Variants—Monomeric fractions of MSA (Calbiochem) and ferase (GST) tag from Schistosoma japonicum into the HSA (Sigma-Aldrich) were purified by size-exclusion chroma- pcDNA3-GST-h2m-oriP vector, which also contains a tography on Superdex 200 (2.6  60 cm, Amersham Bio- cDNA-encoding h2m and the Epstein-Barr virus origin of sciences) operated on a gradient fraction collector (Pharma- replication (oriP) (32). The final vector was sequenced and cia Biotech). The column was loaded with 1.5–5 ml of sample WT denoted pcDNA3-mFcRn -GST-h2m-oriP. at a concentration of 75–100 mg/ml. As elution buffer, FEBRUARY 12, 2010• VOLUME 285 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4827 Cross-species Binding to FcRn 0.05 M Tris, 0.2 M NaCl, 2 mM EDTA, 0.02% NaN was used, size-exclusion chromatography analysis on an analytical and the mixture was filtrated through a 0.22-m filter prior Superdex 200 (1  30 cm) operated on an LKB high-perfor- to use. The purity of the collected fractions was tested by mance liquid chromatograph equipped with a Titan pump and eluted at 0.3 ml/min. ELISA—Microtiter wells (Nunc) were coated with 100 lof bovine serum albumin-NIP at 1 g/ml, incubated overnight at 4 °C, and washed three times with PBS/0.005% Tween 20 (PBS/ T), pH 7.4. They were then blocked with 4% skimmed milk (Acumedia) for1hat room temperature and washed as above. Serial dilutions (1 g/ml to 0.0004 g/ml) of anti-NIP hIgG1, H435A hIgG1 , mIgG1, and mIgG2b were added for 1 h at room temperature and washed with PBS/T, pH 6.0 or pH 7.4. 1 g/ml GST-tagged smFcRn or shFcRn variants preincubated with a horseradish peroxidase-conjugated goat anti-GST antibody (Amersham Biosciences) were added for1hat room tempera- ture followed by washing with PBS/T, pH 6.0, or PBS/T, pH 7.4. Binding was visualized using tetramethylbenzidine substrate (Calbiochem). Binding to MSA or HSA was performed using serial dilutions of albumin (200 g/ml to 0.010 g/ml) coated in microtiter wells. The following steps were as described above. SPR Analyses—SPR analyses were performed on a BIAcore 3000 instrument (Amersham Biosciences) using CM5 chips, and immobilization of smFcRn-GST and shFcRn-GST variants or smFcRn (kind gift from Dr. Sally Ward, University of Texas FIGURE 1. SDS-PAGE analyses of soluble receptor preparations. Secreted WT WT GST-tagged smFcRn and shFcRn molecules were purified from superna- Southwestern Medical Center, Dallas, TX) was performed tants harvested from transiently transfected HEK 293E cells and analyses by using the amine coupling kit (Amersham Biosciences). Protein 12% SDS-PAGE. Lane 1 shows protein standard. Lanes 2 and 3 show non- WT reduced (NR) and reduced (R) samples of shFcRn , respectively. Lanes 4 and samples (10 g/ml) were injected in 10 mM sodium acetate at WT 5 show NR and R samples of smFcRn . The bands corresponding to GST pH 4.5 (Amersham Biosciences), all as described by the manu- fused HCs and h2m are indicated by arrows. facturer. Unreacted moieties on the surface were blocked with 1 M ethanolamine. For all experi- ments, phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 6.0 or 7.4, or HBS-P buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% surfactant P20) at pH 7.4 were used as running buffer or dilution buffer. Kinetic measurements were performed using a low density immobilized surface (100–200 resonance units (RU)). Serial dilutions of hIgG1 (2000.0–31.2 nM), mIgG1 (1000.0– 15.6 nM), MSA (20.0–0.3 M), and HSA (200.0–3.1 M) were injected at pH 6.0 or 7.4, at a flow rate of 50 l/min at 25 °C. Additive binding was recorded by injecting HSA (10 M), MSA (5 M), hIgG1 (100 nM), or mIgG1 (100 nM) alone or two at a time at 25 °C at 20 l/min at pH 6.0 over immobilized shFcRn (600 RU) or smFcRn (600 RU). Com- petitive binding was measured by injecting shFcRn (50 nM) or smFcRn (100 nM) alone or together with dif- WT WT WT FIGURE 2. pH-dependent binding of shFcRn and smFcRn to IgG variants in ELISA. Binding of shFcRn WT H435A WT WT ferent amounts of HSA or MSA (A) and smFcRn (C) to hIgG1, hIgG1 , mIgG1, and mIgG2b at pH 6.0. Binding of shFcRn (B) and smFcRn (D) H435A to hIgG1, hIgG1 , mIgG1, and mIgG2b at pH 7.4. The numbers given represent the mean of triplicates. (10.0–0.05 M) over immobilized 4828 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 7 •FEBRUARY 12, 2010 Cross-species Binding to FcRn acids 1–269) of mFcRn HC was PCR-amplified from a mouse liver cDNA library and found to be iden- tical with published sequences (data not shown). The HC was then expressed as fusion to GST after transient transfection of HEK 293E cells as described before (32). The vector used also carried the h2m cDNA. Harvested cell supernatants were pooled and applied to a GSTrap column for capture of WT chimeric smFcRn -GST mole- cules. SDS-PAGE analyses under non-reducing and reducing condi- tions showed the appearance of two main bands at 75 and 12 kDa that represent the GST-tagged mouse FcRn HC and h2m, respec- tively (Fig. 1). The shFcRn HC pre- pared in the same fashion migrated as a band of 65 kDa, which is in agreement with previous reports of heavier glycosylation of mFcRn than the human form (36, 37). Both receptor fractions contained bands of higher molecular weight, which represent covalent aggregates that resolve under reducing conditions. This is in agreement with previous WT WT FIGURE 3. pH-dependent binding of shFcRn and smFcRn to albumin variants in ELISA. Binding of WT WT reports for other GST fusion mole- shFcRn to HSA (A) and MSA (B) at pH 6.0 and 7.4. Binding of smFcRn to MSA (C) and HSA (D) at pH 6.0 and 7.4. Numbers given represent the mean of triplicates. cules (38, 39). The total amount of WT secreted chimeric smFcRn HSA (2600 RU) or MSA (2000 RU). In all cases, to correct obtained was 0.4 mg/liter supernatant, slightly higher than WT for nonspecific binding and bulk buffer effects, responses that reported for production of shFcRn (32). Thus, mFcRn obtained from the control surfaces and blank injections were HC was shown to assemble with h2m in HEK 293E cells, and subtracted from each interaction curve. Kinetic rate values the heterodimer was secreted as a chimeric receptor. were calculated using predefined models (Langmuir 1:1 ligand Functional Integrity Determined by ELISA—The functional WT model, heterogeneous ligand model, and steady-state affinity integrity of the chimeric smFcRn was confirmed by testing model) provided by using BIAevaluation 4.1 software. The its IgG-binding properties. Binding to mIgG and hIgG variants closeness of the fit, described by the statistical value  , which were investigated and compared side by side with the binding represents the mean square, was lower than 2.0 in all affinity ability of the human counterpart using a pH-dependent ELISA. estimations. Dilutions of anti-NIP IgG variants were captured on NIP-con- Sequence Analyses—ClustalW was used for amino acid jugated bovine serum albumin-coated microtiter wells. GST- sequence alignments. The NCBI accession numbers of the tagged receptors were then added at acidic or physiological pH, FcRn HC sequences: NM_004107 (human), NM_176657 and binding was detected using a horseradish peroxidase-con- (bovine), NM_033351 (rat), and NM_010189 (mouse). For the jugated anti-GST antibody. Fig. 2 (A and B) shows pH-depend- 2m sequences: AAA51811 (human), NP_776318 (bovine), ent binding of shFcRn to hIgG1, whereas a hIgG1 mutant, H435A NP_036644 (rat), and NP_033865 (mouse). hIgG1 , did not interact at either pH. Moreover, mIgG1 did not bind and mIgG2b bound weakly, all in agreement with RESULTS previous findings (30). Repeating the assays under the same WT Preclinical evaluations of novel IgGs, Fc, and albumin fusions conditions showed that the chimeric smFcRn variant inter- are frequently performed in rodents. Thus, in vitro interaction acted with mIgG1 and mIgG2b at acidic pH (Fig. 2C) and only analyses of such constructs regarding cross-species FcRn bind- very weakly at pH 7.4 (Fig. 2D). Binding to hIgG1 was consid- ing may give information valuable when predicting in vivo bio- erably stronger than to the mIgG subclasses, and hIgG1 bound distribution and efficacy. both at acidic and physiological pH (Fig. 2, C and D). Taken Construction and Expression of a Chimeric smFcRn Variant— together, the results are as those previously reported for the A cDNA segment encoding the three ectodomains (amino murine receptor (40, 41), and thus, chimeric GST-tagged FEBRUARY 12, 2010• VOLUME 285 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4829 Cross-species Binding to FcRn WT smFcRn has the same IgG-binding properties as the mouse measurements were run using covalently immobilized receptor receptor counterpart. and injection of IgG or albumin. Dilutions of mIgG1 were WT We next explored the interaction of the soluble receptor vari- injected over CM5 surface of smFcRn , and reversible, con- ants with albumin. Dilutions of monomeric size-exclusion centration-dependent binding was observed at pH 6.0 (Fig. 4A), chromatography isolated MSA and HSA (supplemental Fig. 1) in contrast to almost negligible binding responses at pH 7.4 were coated directly in ELISA wells, and pH-dependent binding (Fig. 4B). The SPR data were fitted to the heterogeneous ligand studies were performed. Fig. 3 (A and B) shows binding of binding model. This model has been used extensively to evalu- WT shFcRn to both HSA and MSA, respectively, but not at phys- ate the FcRn-IgG interaction when FcRn is immobilized (12, 37, iological pH. Thus, shFcRn does not discriminate against bind- 42, 43). The K values obtained were 8.5  0.5  10 M (K ) D D1 WT 9 ing to MSA as it does to mIgGs. smFcRn bound both MSA and 450.0  65.0  10 M (K ). This is in accordance with D2 and HSA, although lower binding responses were obtained values obtained by others with immobilized murine receptor compared with shFcRn binding (Fig. 3, C and D). No detectable (19). binding was seen to either albumin variant at pH 7.4. Cross-species binding to hIgG1 generated responses clearly Determination of Binding Kinetics by SPR Analyses—The stronger than those recorded for mIgG1 (Fig. 4C) and derived expressed receptor domains are normally cell bound and kinetics gave values of 0.1  0.0  10 M (K ) and 63.2 D1 exposed to circulating or pinocytosed soluble ligands. Thus, all 4.8  10 M (K ) at pH 6.0. Thus, a 85-fold decreased K D2 D1 was found compared with that of the smFcRn-mIgG1 interaction. At pH 7.4, significant concentration- dependent and reversible binding responses were obtained (Fig. 4D), and the affinity could be calculated with a K of 10 M. These data D1 are summarized in Table 1. The kinetics of cross-species IgG bind- ing have previously been recorded by SPR with IgG immobilized on the chip (44–46). This receptor:ligand orientation estimates a lower affin- ity for the interaction than that recorded here, where the receptor is immobilized. The trends were obtained, however. To evaluate binding of MSA to WT smFcRn , dilutions of monomeric MSA were injected over the immo- bilized receptor at pH 6.0, and the representative sensorgram demon- strates reversible binding (Fig. 5A). The data fitted well to a simple first-order bimolecular interaction model applied with the BIAevalua- tion software and gave a K of 9.3 WT FIGURE 4. SPR analyses of the smFcRn interaction with hIgG1 and mIgG1. Representative sensorgrams WT 0.4  10 M (Table 2). No binding of serial dilutions of mIgG1 over immobilized smFcRn at pH 6.0 (A) and 7.4 (B), serial dilutions of hIgG1 over WT WT WT immobilized smFcRn at pH 6.0 (C) and 7.4 (D). In all experiments smFcRn was immobilized by amine of MSA to smFcRn was obtained WT coupling to 100 –200 RU. Dilutions of mIgG1 and hIgG1 were injected over an immobilized smFcRn at at pH 7.4 (Fig. 5B). 25 °C. The flow rate was 50 l/min. TABLE 1 WT Kinetics of the IgG interactions with smFcRn a b c c Analyte pH k k k k K f K f a1 d1 a2 d2 D1 1 D2 2 4 4 4 3 10 /Ms 10 /s 10 /Ms 10 /s nM %nM % mIgG1 6.0 16.1  0.6 13.7  0.4 3.8  0.4 17.1  0.5 8.5  0.5 78.9 450.0  65.0 21.1 mIgG1 7.4 ND ND ND ND ND ND ND ND hIgG1 6.0 41.3  0.6 0.5  0.0 7.4  0.2 4.6  0.4 0.1  0.0 82.4 63.2  4.8 17.6 hIgG1 7.4 4.1  0.3 478.5  33.2 1.6  0.1 2.7  0.3 1169.0  0.7 84.4 168.7  30.0 15.6 a WT Dilutions of mIgG1 and hIgG1 were injected over immobilized smFcRn as shown in Fig. 4. The binding measurements were performed at pH 6.0 or 7.4. Fractional occupancies, f and f , of the two independent, parallel interactions. 1 2 The kinetic rate constants were obtained using the heterogeneous ligand binding model, which gave the best global fit using the BIAevaluation 4.1 software. The model assumes two independent, parallel reactions with immobilized smFcRn-GST. The kinetic values represent the average of triplicates. ND, not determined due to no or very low binding responses. 4830 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 7 •FEBRUARY 12, 2010 Cross-species Binding to FcRn WT WT Monomeric HSA bound smFcRn , but very weakly and shFcRn interacted with MSA at pH 6.0 (Fig. 5D) and not at with fast kinetics at pH 6.0 (Fig. 5C), while no binding at pH 7.4 pH 7.4 (supplemental Fig. 3B). The estimated K at pH 6.0 was was observed (supplemental Fig. 3A). Injection of higher con- 0.8 0.2 10 M. The kinetic measurements are summarized centrations of HSA increased binding responses, but aggrega- in Table 2. When comparing kinetics, the dissociation rates tion of HSA obscured the results. However, these data could be were found to differ dramatically and increased in the following fitted to a steady-state binding model and gave rise to an esti- order: smFcRn:MSA  shFcRn:HSA  shFcRn:MSA. No data mated K of 86.2 4.1 10 M. The results are not affected by could be obtained for the smFcRn:HSA pair, due to fast kinetics. WT the chimeric composition of smFcRn, because the same weak Taken together, shFcRn bound more strongly than WT binding responses were obtained using a fully murine form of smFcRn to both albumin species, and MSA bound more FcRn (supplemental Fig. 2). strongly than HSA to both receptor variants. Thus, an affinity hierarchy appears as follows; shFcRn:MSA  shFcRn:HSA smFcRn:MSA  smFcRn:HSA. Cross-species Competitive Bind- ing—To investigate the functional impact of cross-species binding, a constant amount of each receptor was preincubated with titrated amounts of MSA or HSA and injected over immobilized HSA or MSA. The percent inhibition of FcRn binding was calculated in each case. MSA preincubated with WT shFcRn inhibited receptor bind- ing to immobilized HSA more effi- ciently than HSA, because 3-fold more HSA than MSA was required to reach 50% inhibition (0.16 versus 0.05 M) (Fig. 6A). Furthermore, HSA was shown to inhibit WT smFcRn binding to MSA rather poorly, as in this case 10-fold more HSA than MSA was required to reach 50% inhibition (Fig. 6B). Mapping the Differences in Albu- min Binding Properties—Previ- ously, we reported that the con- served His-166, located to the WT WT FIGURE 5. SPR analyses of the shFcRn and smFcRn interaction with HSA and MSA. Representative WT 2-domain of human heavy chain, sensorgrams of serial dilutions of MSA injected over immobilized smFcRn at pH 6.0 (A) and 7.4 (B), serial WT dilutions of HSA injected over immobilized smFcRn at pH 6.0 (C), and serial dilutions of MSA injected to is crucial for binding to HSA, WT WT WT immobilized shFcRn at pH 6.0 (D). In all experiments shFcRn and smFcRn were immobilized by amine because mutation of this residue coupling to500 – 800 RU. Dilutions of MSA and HSA were injected over an immobilized receptor at 25 °C. The flow rate was 50 l/min. to alanine completely eliminate TABLE 2 Kinetics of the albumin interactions with FcRn variants a b Albumin species FcRn species FcRn variant k k K K Req a d D D 3 3 10 /Ms 10 /s M M c d MSA Mouse WT 4.2  0.5 39.4  3.1 9.3  0.4 ND MSA Human WT 3.8  0.0 3.1  0.1 0.8  0.2 ND b e HSA Mouse WT NA NA NA 86.2  4.1 HSA Human WT 2.7  1.3 12.2  5.9 4.5  0.1 4.6  0.5 HSA Mouse L166R/G167E NA NA NA 26.8  0.1 MSA Human R164L/E165G 0.7  0.1 3.4  0.1 4.8  0.1 ND MSA Mouse L166R/G167E 2.7  0.2 18.5  0.5 6.8  1.8 ND HSA Human R164L/E165G 3.2  0.1 26.3  0.2 8.2  0.1 ND Dilutions of MSA and HSA were injected over immobilized receptor as shown in Figs. 5 and 8. The steady-state affinity constant was obtained using an equilibrium (Req) binding model supplied by the BIAevaluation 4.1 software. The kinetic values represent the average of triplicates. The kinetic rate constants were obtained using a simple first-order (1:1) bimolecular interaction model. ND, not determined. NA, not acquired because of fast kinetics. The kinetic values have been published in Ref. 10. FEBRUARY 12, 2010• VOLUME 285 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4831 Cross-species Binding to FcRn residues in ligand binding, Arg-164 and Glu-165 were mutated to leucine and glycine in shFcRn, whereas Leu-166 and Gly-167 were mutated to arginine and glutamic acid in smFcRn. Titrated amounts of monomeric HSA and MSA were again injected over immobilized receptor variants at pH 6.0. Rep- resentative sensorgrams demonstrate reversible binding responses at acidic pH, and the calculated binding kinetic val- ues differ from that of the wild types (Fig. 8, A–D, and Table 2). L166R/G167E The humanized smFcRn variant bound HSA 3-fold more strongly than the wild-type mouse form, and the binding affinity for MSA was also slightly increased. Further- R164L/E165G more, rodentized shFcRn bound HSA with an affin- ity of 2-fold weaker and MSA with a 6-fold weaker affinity WT than shFcRn . Thus, exchange of human-mouse amino acids in the vicinity of the key histidine residue decreased the differ- ences in albumin-binding properties at acidic pH. However, the R164L/E165G affinity of rodentized shFcRn was not completely WT reduced to that of smFcRn , and the affinity of humanized L166R/G167E smFcRn did not totally reach the binding affinity of WT shFcRn . Also, the impact of the mutations on hIgG1 binding was investigated by ELISA. No differences in binding were detected H166A R164L/E165G for shFcRn and shFcRn compared with WT shFcRn (Fig. 8E). Mutation of two conserved glutamic acids, Glu-115 and Glu-116 (highlighted in Fig. 7B), to alanines E115A/E116A (shFcRn ), completely eliminated binding to hIgG1 (Fig. 8E). This result supports a key role for these negatively charged residues in IgG binding, as previously shown by others H168A L166R/G167E (30, 47). Both smFcRn and smFcRn bound hIgG1 like the wild-type receptor (Fig. 8F). Thus, mutation of amino acids close to the conserved histidine did not influence binding to hIgG1. Bifunctional FcRn Ligand Binding—shFcRn has been shown FIGURE 6. Competitive FcRn-albumin binding across species. A, serial dilu- to bind both hIgG and HSA simultaneously in a pH-depen- tions of HSA (0.5– 0.05 M) and MSA (0.5– 0.05 M) were preincubated with WT shFcRn (0.05 M) and injected over immobilized HSA (2600 RU). B, serial dent manner (11). Cross-species binding of both ligands to dilutions of HSA (10.0 – 0.1M) and MSA (1.0 – 0.1M) were preincubated with FcRn may reveal how they are transported and protected WT smFcRn (0.10 M) and injected over immobilized MSA (2000 RU). The from degradation in WT and transgenic mouse strains. We representative binding data are presented as percent inhibition of the FcRn binding to immobilized albumin. Injections were performed at 25 °C, and the investigated the effect of each ligand on the binding of the flow rate was 50 l/min. other by injecting IgG and albumin, from both species, sep- arately or together as a preincubated sample, over surfaces binding to HSA at acidic pH, whereas binding to hIgG is immobilized with smFcRn or shFcRn at acidic pH. Fig. 9 (A retained (10). This residue is conserved in all known FcRn and B) shows the resulting responses for binding to shFcRn sequences, including the mouse and rat HCs (Fig. 7A) (10). and smFcRn, respectively. Both receptors bound their native Mutation to alanine of the corresponding residue in the mouse ligands in an independent and additive manner. shFcRn counterpart (His-168) completely eliminated binding to HSA ignored binding to mIgG, and smFcRn bound very weakly to (100 M), and only weak binding was detected for MSA (20 M) HSA. In all cases, however, for both receptors, neither ligand when injected over a high density surface with immobilized (IgG or albumin from both species) interfered with binding mutant at acidic pH (supplemental Fig. 4, A and B). of the other. We speculated whether species differences in binding kinet- DISCUSSION ics may be caused by non-conserved amino acids found in prox- imity to His-166 in the folded molecule. Interestingly, inspec- Proper folding and cellular transport of the FcRn HC is abso- tion of the flanking amino acids revealed major non-conserved lutely dependent on association with 2m in the endoplasmic variations, because the neighboring exposed basic Arg-164 and reticulum (48). Thus, both polypeptides need to be present for acidic Glu-165 in humans are replaced by the hydrophobic generation and secretion of cell bound as well as truncated leucine and glycine residues in rodents, respectively (Fig. 7, A forms of heterodimeric FcRn. Expression of functional chi- and B). His-166 and the non-conserved amino acid residues meric FcRn has earlier been demonstrated in vivo in mice trans- (Arg-164 and Glu-165) are highlighted in the human crystal genic for the hFcRn HC (4) or the bovine FcRn HC (49). In both structure shown in Fig. 7B. To explore the putative role of these cases, the HC associates with mouse 2m into a functional 4832 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 7 •FEBRUARY 12, 2010 Cross-species Binding to FcRn injected the receptor (45, 46, 50). In this situation, the actual affinities that were calculated were lower than those obtained here using the hetero- geneous ligand binding model. However, the binding hierarchies WT were the same, with shFcRn WT ignoring mIgG and smFcRn binding better to hIgG than to mIgG. The high affinity is in agree- ment with binding studies of FcRn expressed on cells (51). Importantly, with IgG immobi- lized, the interaction between WT smFcRn and hIgG at physiologi- cal pH was barely detectable. By immobilizing the receptor, we were able to obtain kinetic data that sug- gest a difference in K of four logs D1 for the interactions at acidic and physiological pH. The higher affinity of hIgG1 for WT smFcRn compared with the smFcRn-mIgG interaction at pH 6.0 may indicate that half-life in WT mice could be overestimated. How- ever, the fact that hIgG1 also binds with reasonable affinity at physio- logical pH could counteract the effect, because it has been shown that such interaction lowers the FIGURE 7. The crystal structure of shFcRn. A, amino acids flanking His-166 (hFcRn) and His-168 (mFcRn) half-life (52). In any case, half-life located within the heavy chain 2-domain are shown. His-166 and His-168 are shown in bold. The non-con- estimations of mIgG and hIgG in served Arg-164 and Glu-165 of hFcRn, and Leu-167 and Gly-168 of mFcRn are shown in italic. B, the crystal structure of shFcRn shown in two orientations. The localization of amino acids essential for IgG (Glu-115 and WT mice show approximately the Glu-116) and albumin (His-166) binding are highlighted as blue and red spherical balls. The non-conserved same values (17). Arg-164 and Glu-165 (human) are highlighted with yellow and gray spherical balls, respectively. The FcRn heavy chains are shown in green and the 2m in orange. The figures were designed using PyMOL (DeLano Scientific) SPR analyses showed that WT with the crystallographic data of shFcRn (37). smFcRn interacted pH depen- dently with MSA with an estimated transmembrane-anchored chimeric FcRn. However, direct K of 9.3  0.4  10 M at acid pH. This is the first report on interaction studies of soluble forms of chimeric FcRn molecules in vitro kinetics of the smFcRn-MSA interaction, a finding that with ligands have not been reported. supports the role of FcRn in albumin half-life regulation in mice In this study, we show that a truncated mFcRn HC assembles (3, 53). with h2m, and that the heterodimer is secreted from HEK The remarkably long half-life of albumin was well recognized 293E cells. The functional integrity of the chimeric smFcRn was before its relationship with FcRn was discovered and utilized to extensively investigated by ELISA analyses and revealed bind- enhance the in vivo effect of short-lived therapeutic substances. ing to mIgG1, mIgG2b, hIgG1 and no detectable binding to a For instance, HSA-fused interferon 2b is now undergoing Phase H435A hIgG1 mutant. The chimeric receptor performed as a 3 trials (54), and other HSA fusions are under study. Importantly, completely murine receptor and was then utilized in a series of such constructs require animal models for preclinical evaluation. ELISA and SPR experiments to obtain new information about Recent reports have addressed the in vivo half-life of HSA cross-species ligand binding to FcRn. fused or targeted molecules in mice (25, 28) and argued that the The amino acid sequences of bovine, rat, and mouse 2m increase in half-life observed is a consequence of FcRn-medi- showed 73%, 68, and 66% homology with the human counterpart, ated rescue. Improved tumor imaging in rodents has been and the corresponding values for the FcRn HC were 76%, 64 and obtained using antitumor antigen antibody fragments geneti- 66%, respectively (supplemental Table 2 and 3). Thus, FcRn HC cally fused to HSA- or HSA-binding proteins (26, 29, 55). How- from other species may well be co-expressed with h2m in HEK ever, no complementary and comparative studies of such 293E cells to produce a variety of chimeric FcRn variants. mFcRn cross-species binding to HSA have been reported. Here, To perform SPR, the receptors were immobilized on the chip, we demonstrate a large difference in the kinetics of albumin WT and the ligands were injected. Others have immobilized IgG and binding to the mouse and human forms of FcRn. smFcRn FEBRUARY 12, 2010• VOLUME 285 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4833 Cross-species Binding to FcRn ther molecule shows more than half the serum persistence of endoge- nous rat albumin (56). Notably, mouse and rat FcRn HCs showed high homology (89%) (supplemen- tal Table 3), as did rat and mouse albumin sequences (90%). Thus, the rat FcRn-HSA interaction is likely as weak as the smFcRn-HSA interac- tion. Albumin-targeted molecules have been described that achieve the same half-life as endogenous albumin. This is the case with human domain antibodies selected to bind albumin (57). Two anti-rat albumin domain antibodies with low (1 M) and high (13 nM) affinity showed half-lives in rats of 43 and 53 h, respectively. Rat albumin has a half-life of 53 h, similar to the high affinity domain antibody. WT Although shFcRn ignores mIgG, it interacts strongly with MSA. The affinity for MSA was 100- fold stronger than that of the murine receptor. Based on this, one would predict that the mouse strain transgenic for the hFcRn HC would bind strongly to endog- enous MSA and protect it from degradation. This was indeed the case, and a 46% increase of the MSA levels in such mice has been observed (3). The presence of MSA bound to the human receptor, and also high serum concentrations of MSA, will surely affect rescue of HSA-associ- ated molecules that compete for the same binding site on the receptor. FIGURE 8. SPR analyses of albumin binding to rodentized and humanized FcRn variants. Representative R164L/E165G sensorgrams of serial dilutions of HSA (A) and MSA (B) injected over immobilized rodentized shFcRn Notably, the off rate of MSA is 10 L166R/G167E at pH 6.0. Serial dilutions of MSA (C) and HSA (D) injected over smFcRn . In all experiments the receptor times lower than that of HSA. variants were immobilized by amine coupling to 1000–2000 RU. Dilutions of MSA and HSA were injected over WT H166A immobilized receptors at 25 °C. The flow rate was 50 l/min. E, binding of shFcRn , shFcRn , Transgenic mice, fortified with R164L/E165G E115A/E116A WT H166A shFcRn , and shFcRn to hIgG1 at pH 6.0 in ELISA. F, binding of smFcRn , smFcRn , and serum hIgG, are useful when evalu- L166R/G167E smFcRn to hIgG1 at pH 6.0 in ELISA. The numbers given represent the mean of triplicates. ating serum persistence of engi- binds MSA with a K of 10 M. The affinity for the endoge- neered hIgGs, but the half-life of nous ligand is 10-fold higher than that for HSA, a fact that HSA variants and conjugates may well be underestimated. The WT nicely correlates with the inhibition data where smFcRn was latter is supported by the competitive data presented here WT shown to prefer MSA over HSA. This must necessarily affect where MSA efficiently inhibited shFcRn binding to HSA. the in vivo half-life of both HSA and HSA fused molecules in The binding sites for hIgG and HSA are distally localized mice in the presence of high amounts of circulating endoge- in the 2-domain of the hFcRn HC (10). In line with this, we nous albumin. When HSA-fused molecules show a moderate show that all combinations of ligands bound additively to WT increase in half-life in rodents, and not an extended half-life both receptor forms, and that shFcRn ignores mIgG while similar to that of endogenous albumin, it may simply be an binding strongly to MSA. 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Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding 2

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American Society for Biochemistry and Molecular Biology
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0021-9258
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10.1074/jbc.m109.081828
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 7, pp. 4826 –4836, February 12, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in □ S Immunoglobulin G and Albumin Binding Received for publication, November 4, 2009, and in revised form, December 3, 2009 Published, JBC Papers in Press, December 14, 2009, DOI 10.1074/jbc.M109.081828 ‡1 ‡ ‡ §¶ ‡2 Jan Terje Andersen , Muluneh Bekele Daba , Gøril Berntzen , Terje E. Michaelsen , and Inger Sandlie ‡ § From the Department of Molecular Biosciences and Centre for Immune Regulation and Institute of Pharmacy, University of Oslo, P.O. Box 1041, N-0371 Oslo and the Norwegian Institute of Public Health, N-0403 Oslo, Norway The neonatal Fc receptor (FcRn) regulates the serum half-life half-life, transport of IgG across intestinal epithelia and pla- of both IgG and albumin through a pH-dependent mechanism centa, as well as enhancement of neutrophil phagocytosis of that involves salvage from intracellular degradation. Therapeu- immune complexes, as reviewed previously (1). Moreover, the tics and diagnostics built on IgG, Fc, and albumin fusions are receptor plays a role in antibody-mediated antigen presenta- frequently evaluated in rodents regarding biodistribution and tion by dendritic cells (2). FcRn has also been found to salvage pharmacokinetics. Thus, it is important to address cross-species albumin from intracellular degradation (3), in a fashion similar ligand reactivity with FcRn, because in vivo testing of such mol- to that described for IgG, which involves receptor ligand inter- ecules is done in the presence of competing murine ligands, both actions in acidified endosomal compartments (1). Hence, FcRn in wild type (WT) and human FcRn (hFcRn) transgenic mice. affects diverse and important immunological and non-immu- Here, binding studies were performed in vitro using enzyme- nological processes. linked immunosorbent assay and surface plasmon resonance FcRn is a heterodimeric receptor consisting of a transmem- WT with recombinant soluble forms of human (shFcRn ) and brane heavy chain (HC) that is non-covalently associated with WT mouse (smFcRn ) receptors. No binding of albumin from 2-microglobulin (2m). Consequently, the significance of either species was observed at physiological pH to either recep- FcRn has been extensively documented in knockout mouse tor. At acidic pH, a 100-fold difference in binding affinity was models lacking 2m or the HC. Such deficient mice have IgG WT observed. Specifically, smFcRn bound human serum albumin serum levels of 20–30% that of wild-type mice and a 60% WT with a K of 90 M, whereas shFcRn bound mouse serum reduced level of MSA (3, 4). A human example is the rare famil- WT albumin with a K of 0.8 M. shFcRn ignored mouse IgG1, ial hypercatabolic hypoproteinemia syndrome that is charac- WT and smFcRn bound strongly to human IgG1. The latter pair terized by reduced serum levels of both hIgG and HSA (5). An also interacted at physiological pH with calculated affinity in the explanation was provided when deficient FcRn expression was micromolar range. In all cases, binding of albumin and IgG from demonstrated as a result of a point mutation in the 2m-encod- either species to both receptors were additive. Cross-species ing gene sequence that disrupts efficient secretion (6). Thus, albumin binding differences could partly be explained by non- FcRn is truly bifunctional and contributes to maintaining the conserved amino acids found within the 2-domain of the high levels of IgG as well as albumin in serum, with levels receptor. Such distinct cross-species FcRn binding differences amounting to 12 and 40 mg/ml, respectively, in mice and must be taken into consideration when IgG- and albumin-based humans. therapeutics and diagnostics are evaluated in rodents for their The FcRn HC consists of three ectodomains (1, 2, and 3), pharmacokinetics. a short transmembrane region, and a cytoplasmic tail (1). Mutagenesis and crystallographic studies have uncovered that the FcRn-IgG interaction is mediated by Fc-localized residues, The major histocompatibility class I-related neonatal Fc especially Ile-253, His-310, and His-435, and acidic surface- receptor (FcRn) is a versatile receptor that regulates serum IgG exposed residues on the 2-domain of the HC (7–9). The inter- action is strictly pH-dependent with binding at acidic pH and * This work was supported by grants from the Steering Board for Research in no or very weak binding at physiological pH. The histidines are Molecular Biology, Biotechnology and Bioinformatics at the University of mainly responsible for the pH dependence, because they are Oslo (to J. T. A.), The Norwegian Research Council (Grant 179573), and the Norwegian Cancer Society (Grant B95078). protonated under acidic conditions. Although the FcRn-albu- □ S The on-line version of this article (available at http://www.jbc.org) contains min interaction is less well characterized, data indicate that supplemental Figs. 1– 4 and Tables 1–3. domain III of albumin binds to the HC 2-domain at a site To whom correspondence may be addressed. Tel.: 47-22-85-47-93; Fax: 47-22-85-40-61; E-mail: [email protected]. distant from the IgG binding site, because His-166 is crucial for To whom correspondence may be addressed. E-mail: [email protected]. the interaction (10, 11). Thus, both ligands may bind simulta- The abbreviations used are: FcRn, neonatal Fc receptor; HC, heavy chain; HEK, neously in a pH-dependent manner. humanembryonickidney;hFcRn,humanFcRn;hIgG,humanIgG;HSA,human serum albumin; h2m, human 2-microglobulin; mIgG, mouse IgG; MSA, Knowledge of FcRn-IgG biology explains the prolonged half- mouse serum albumin; oriP, origin of replication; RU, resonance unit; shFcRn, life of IgG Fc-fused therapeutics (1, 4, 12, 13). Understanding of soluble hFcRn; smFcRn, soluble mouse FcRn; SPR, surface plasmon resonance; the FcRn-IgG interaction at the atomic level has prompted the WT, wild type; NIP, 3-iodo-4-hydroxy-5-nitrophenacetyl; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase. development of novel IgG-based therapeutics with point muta- 4826 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 7 •FEBRUARY 12, 2010 This is an Open Access article under the CC BY license. Cross-species Binding to FcRn tions in their Fc part that modulate serum half-life (14–19). Construction of Mutant FcRn Variants—A single amino acid- Furthermore, improved half-life and efficiency of a number of substituted mFcRn variant was constructed by mutating His- 168 to alanine by site-directed mutagenesis using the plasmid small therapeutic molecules and proteins that are normally WT pcDNA3-mFcRn -GST-h2m-oriP and the primers cleared rapidly from the circulation have been achieved by strategies such as chemical conjugation or genetic fusion to mFcRnH168AForw and mFcRnH168ARev. Three double mutant E117A/E118A R164L/E165G albumin itself (20–25) or any of several albumin binding mol- FcRn variants, named hFcRn , hFcRn , and L166R/G168E mFcRn , were constructed using the templates ecules (26–29). WT WT pcDNA3-mFcRn -GST-h2m-oriP and pcDNA3-hFcRn - Mice are routinely used as convenient first line models for GST-h2m-oriP. The primer sequences used are all listed in preclinical evaluation of such therapeutics. Thus, it is crucial to supplemental Table 1. understand if and how mFcRn interacts with human ligands. Expression and Purification of Soluble FcRn Variants—For Indeed, mFcRn has been shown to be rather promiscuous in its transient transfections, the hFcRn- and mFcRn-encoding plas- binding to IgG. It binds IgG from different species, including mids were transfected into HEK 293E cells (ATCC) using Lipo- hIgG. On the other hand, hFcRn discriminates binding to mIgG fectamine 2000 (Invitrogen) following the manufacturer’s (except for weak binding to mIgG2b) (30). This latter finding instructions. HEK 293E cells were cultured in Dulbecco’s mod- has greatly contributed to the understanding of the fast clear- ified Eagle’s medium (BioWhittaker) using standard condi- ance and disappointing therapeutic effects obtained using tions. Pooled media were filtrated and applied on a GSTrap FF monoclonal mIgGs in human trials. However, fast or interme- column (5 ml, Amersham Biosciences) connected to a semiau- diate clearance can also be favorable, as demonstrated for IgG tomatic workstation and recorder, and purifications were per- immunoconjugates approved for cancer imaging and therapy formed essentially as recommended in the manufacturer’s (16, 31). manual. Eluted fractions were pooled, concentrated, and ana- Mice have recently been constructed that lack the mFcRn lyzed under non-reducing or reducing condition using -mer- HC and are transgenic for the human counterpart (4). Such captoethanol (Sigma-Aldrich). Samples of 2 g of each receptor mice express hFcRn that is exposed to the murine ligands, were applied on a 12% SDS-PAGE (Bio-Rad). Protein concen- mIgG and MSA. Interestingly, they are found to be unable to trations were determined using a NanoDrop N-1000 spectro- protect mIgG from degradation. Nothing is known about the photometer (NanoDrop Technologies). cross-species interaction between FcRn and albumin. Construction, Production, and Purification of IgG Variants— Herein, we report on important cross-species ligand-FcRn WT A mouse plasmacytoma cell line producing chimeric human binding differences. Specifically, at acidic pH shFcRn binds IgG1 (hIgG1) anti-3-iodo-4-hydroxy-5-nitrophenacetyl (NIP) MSA strongly while ignoring mIgG binding, whereas WT was a gift from Dr. M. Neuberger (Medical Research Council smFcRn binds hIgG1 strongly and HSA very weakly. The Laboratory of Molecular Biology, Cambridge, UK). The con- cross-species differences in albumin binding could partly be struction of this antibody has been described before (33). Pure explained by non-conserved amino acid variations found in the preparations of anti-NIP mIgG1 and mIgG2b were gifts from vicinity of the conserved His-166 of the HC. In vivo, the conse- WT Dr. Gregory Winter (Centre for Protein Engineering, Medical quences of weak binding of HSA to smFcRn may facilitate Research Council Centre, UK). A single amino acid-substituted rapid clearance in the presence of high amounts of endogenous chimeric hIgG1 variant was constructed by mutating His-435 MSA. Such cross-species kinetic differences have great rele- (numbering according to the EU index) to alanine by site-di- vance for preclinical pharmacokinetics and biodistribution rected mutagenesis using the primers hIgG1H435Aforw and evaluations of engineered therapeutic and diagnostic IgGs, Fc, hIgG1H435Arev (listed in supplemental Table 1) and the tem- and albumin fusions in rodents. plate vector pLNOH2/C1 (34), which contains the gene frag- ment encoding the constant HC of hIgG1. The mutant vector EXPERIMENTAL PROCEDURES H435A denoted pLNOH-hIgG1 was transiently expressed in PCR and Subcloning—The cDNA segments encoding trun- HEK 293E cells by co-transfection with the pLNOK vector cated hFcRn HC and human 2m (h2m) were PCR-amplified encoding the mouse lambda light chain as above. Chimeric H435A from a U937 cell line (ATCC) cDNA library followed by sub- hIgG1 was purified on NIP-coupled Sepharose as previ- cloning of the fragments into the pcDNA3-GST vector, all as ously described (35). The integrity of expressed protein was previously described (32). A mouse liver cDNA library (Zyagen) verified by non-reducing SDS-PAGE analyses followed by was used to PCR-amplify a cDNA encoding a truncated version Western blotting using a horseradish peroxidase-conjugated of the mFcRn HC (encoding the endogenous native leader polyclonal rabbit anti-human Fc (Amersham Biosciences) and sequence, 1, 2, and 3 domains; 293 amino acids) using the horseradish peroxidase-conjugated anti-murine lambda light primers mFcRnForw and mFcRnRev, listed in supplemental chain (Southern Biotech) (data not shown). Table 1. Primers were designed to allow in-frame ligation of the Size-exclusion Chromatography Purification of Albumin fragment upstream of a cDNA encoding a glutathione S-trans- Variants—Monomeric fractions of MSA (Calbiochem) and ferase (GST) tag from Schistosoma japonicum into the HSA (Sigma-Aldrich) were purified by size-exclusion chroma- pcDNA3-GST-h2m-oriP vector, which also contains a tography on Superdex 200 (2.6  60 cm, Amersham Bio- cDNA-encoding h2m and the Epstein-Barr virus origin of sciences) operated on a gradient fraction collector (Pharma- replication (oriP) (32). The final vector was sequenced and cia Biotech). The column was loaded with 1.5–5 ml of sample WT denoted pcDNA3-mFcRn -GST-h2m-oriP. at a concentration of 75–100 mg/ml. As elution buffer, FEBRUARY 12, 2010• VOLUME 285 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4827 Cross-species Binding to FcRn 0.05 M Tris, 0.2 M NaCl, 2 mM EDTA, 0.02% NaN was used, size-exclusion chromatography analysis on an analytical and the mixture was filtrated through a 0.22-m filter prior Superdex 200 (1  30 cm) operated on an LKB high-perfor- to use. The purity of the collected fractions was tested by mance liquid chromatograph equipped with a Titan pump and eluted at 0.3 ml/min. ELISA—Microtiter wells (Nunc) were coated with 100 lof bovine serum albumin-NIP at 1 g/ml, incubated overnight at 4 °C, and washed three times with PBS/0.005% Tween 20 (PBS/ T), pH 7.4. They were then blocked with 4% skimmed milk (Acumedia) for1hat room temperature and washed as above. Serial dilutions (1 g/ml to 0.0004 g/ml) of anti-NIP hIgG1, H435A hIgG1 , mIgG1, and mIgG2b were added for 1 h at room temperature and washed with PBS/T, pH 6.0 or pH 7.4. 1 g/ml GST-tagged smFcRn or shFcRn variants preincubated with a horseradish peroxidase-conjugated goat anti-GST antibody (Amersham Biosciences) were added for1hat room tempera- ture followed by washing with PBS/T, pH 6.0, or PBS/T, pH 7.4. Binding was visualized using tetramethylbenzidine substrate (Calbiochem). Binding to MSA or HSA was performed using serial dilutions of albumin (200 g/ml to 0.010 g/ml) coated in microtiter wells. The following steps were as described above. SPR Analyses—SPR analyses were performed on a BIAcore 3000 instrument (Amersham Biosciences) using CM5 chips, and immobilization of smFcRn-GST and shFcRn-GST variants or smFcRn (kind gift from Dr. Sally Ward, University of Texas FIGURE 1. SDS-PAGE analyses of soluble receptor preparations. Secreted WT WT GST-tagged smFcRn and shFcRn molecules were purified from superna- Southwestern Medical Center, Dallas, TX) was performed tants harvested from transiently transfected HEK 293E cells and analyses by using the amine coupling kit (Amersham Biosciences). Protein 12% SDS-PAGE. Lane 1 shows protein standard. Lanes 2 and 3 show non- WT reduced (NR) and reduced (R) samples of shFcRn , respectively. Lanes 4 and samples (10 g/ml) were injected in 10 mM sodium acetate at WT 5 show NR and R samples of smFcRn . The bands corresponding to GST pH 4.5 (Amersham Biosciences), all as described by the manu- fused HCs and h2m are indicated by arrows. facturer. Unreacted moieties on the surface were blocked with 1 M ethanolamine. For all experi- ments, phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 6.0 or 7.4, or HBS-P buffer (0.01 M HEPES, 0.15 M NaCl, 0.005% surfactant P20) at pH 7.4 were used as running buffer or dilution buffer. Kinetic measurements were performed using a low density immobilized surface (100–200 resonance units (RU)). Serial dilutions of hIgG1 (2000.0–31.2 nM), mIgG1 (1000.0– 15.6 nM), MSA (20.0–0.3 M), and HSA (200.0–3.1 M) were injected at pH 6.0 or 7.4, at a flow rate of 50 l/min at 25 °C. Additive binding was recorded by injecting HSA (10 M), MSA (5 M), hIgG1 (100 nM), or mIgG1 (100 nM) alone or two at a time at 25 °C at 20 l/min at pH 6.0 over immobilized shFcRn (600 RU) or smFcRn (600 RU). Com- petitive binding was measured by injecting shFcRn (50 nM) or smFcRn (100 nM) alone or together with dif- WT WT WT FIGURE 2. pH-dependent binding of shFcRn and smFcRn to IgG variants in ELISA. Binding of shFcRn WT H435A WT WT ferent amounts of HSA or MSA (A) and smFcRn (C) to hIgG1, hIgG1 , mIgG1, and mIgG2b at pH 6.0. Binding of shFcRn (B) and smFcRn (D) H435A to hIgG1, hIgG1 , mIgG1, and mIgG2b at pH 7.4. The numbers given represent the mean of triplicates. (10.0–0.05 M) over immobilized 4828 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 7 •FEBRUARY 12, 2010 Cross-species Binding to FcRn acids 1–269) of mFcRn HC was PCR-amplified from a mouse liver cDNA library and found to be iden- tical with published sequences (data not shown). The HC was then expressed as fusion to GST after transient transfection of HEK 293E cells as described before (32). The vector used also carried the h2m cDNA. Harvested cell supernatants were pooled and applied to a GSTrap column for capture of WT chimeric smFcRn -GST mole- cules. SDS-PAGE analyses under non-reducing and reducing condi- tions showed the appearance of two main bands at 75 and 12 kDa that represent the GST-tagged mouse FcRn HC and h2m, respec- tively (Fig. 1). The shFcRn HC pre- pared in the same fashion migrated as a band of 65 kDa, which is in agreement with previous reports of heavier glycosylation of mFcRn than the human form (36, 37). Both receptor fractions contained bands of higher molecular weight, which represent covalent aggregates that resolve under reducing conditions. This is in agreement with previous WT WT FIGURE 3. pH-dependent binding of shFcRn and smFcRn to albumin variants in ELISA. Binding of WT WT reports for other GST fusion mole- shFcRn to HSA (A) and MSA (B) at pH 6.0 and 7.4. Binding of smFcRn to MSA (C) and HSA (D) at pH 6.0 and 7.4. Numbers given represent the mean of triplicates. cules (38, 39). The total amount of WT secreted chimeric smFcRn HSA (2600 RU) or MSA (2000 RU). In all cases, to correct obtained was 0.4 mg/liter supernatant, slightly higher than WT for nonspecific binding and bulk buffer effects, responses that reported for production of shFcRn (32). Thus, mFcRn obtained from the control surfaces and blank injections were HC was shown to assemble with h2m in HEK 293E cells, and subtracted from each interaction curve. Kinetic rate values the heterodimer was secreted as a chimeric receptor. were calculated using predefined models (Langmuir 1:1 ligand Functional Integrity Determined by ELISA—The functional WT model, heterogeneous ligand model, and steady-state affinity integrity of the chimeric smFcRn was confirmed by testing model) provided by using BIAevaluation 4.1 software. The its IgG-binding properties. Binding to mIgG and hIgG variants closeness of the fit, described by the statistical value  , which were investigated and compared side by side with the binding represents the mean square, was lower than 2.0 in all affinity ability of the human counterpart using a pH-dependent ELISA. estimations. Dilutions of anti-NIP IgG variants were captured on NIP-con- Sequence Analyses—ClustalW was used for amino acid jugated bovine serum albumin-coated microtiter wells. GST- sequence alignments. The NCBI accession numbers of the tagged receptors were then added at acidic or physiological pH, FcRn HC sequences: NM_004107 (human), NM_176657 and binding was detected using a horseradish peroxidase-con- (bovine), NM_033351 (rat), and NM_010189 (mouse). For the jugated anti-GST antibody. Fig. 2 (A and B) shows pH-depend- 2m sequences: AAA51811 (human), NP_776318 (bovine), ent binding of shFcRn to hIgG1, whereas a hIgG1 mutant, H435A NP_036644 (rat), and NP_033865 (mouse). hIgG1 , did not interact at either pH. Moreover, mIgG1 did not bind and mIgG2b bound weakly, all in agreement with RESULTS previous findings (30). Repeating the assays under the same WT Preclinical evaluations of novel IgGs, Fc, and albumin fusions conditions showed that the chimeric smFcRn variant inter- are frequently performed in rodents. Thus, in vitro interaction acted with mIgG1 and mIgG2b at acidic pH (Fig. 2C) and only analyses of such constructs regarding cross-species FcRn bind- very weakly at pH 7.4 (Fig. 2D). Binding to hIgG1 was consid- ing may give information valuable when predicting in vivo bio- erably stronger than to the mIgG subclasses, and hIgG1 bound distribution and efficacy. both at acidic and physiological pH (Fig. 2, C and D). Taken Construction and Expression of a Chimeric smFcRn Variant— together, the results are as those previously reported for the A cDNA segment encoding the three ectodomains (amino murine receptor (40, 41), and thus, chimeric GST-tagged FEBRUARY 12, 2010• VOLUME 285 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4829 Cross-species Binding to FcRn WT smFcRn has the same IgG-binding properties as the mouse measurements were run using covalently immobilized receptor receptor counterpart. and injection of IgG or albumin. Dilutions of mIgG1 were WT We next explored the interaction of the soluble receptor vari- injected over CM5 surface of smFcRn , and reversible, con- ants with albumin. Dilutions of monomeric size-exclusion centration-dependent binding was observed at pH 6.0 (Fig. 4A), chromatography isolated MSA and HSA (supplemental Fig. 1) in contrast to almost negligible binding responses at pH 7.4 were coated directly in ELISA wells, and pH-dependent binding (Fig. 4B). The SPR data were fitted to the heterogeneous ligand studies were performed. Fig. 3 (A and B) shows binding of binding model. This model has been used extensively to evalu- WT shFcRn to both HSA and MSA, respectively, but not at phys- ate the FcRn-IgG interaction when FcRn is immobilized (12, 37, iological pH. Thus, shFcRn does not discriminate against bind- 42, 43). The K values obtained were 8.5  0.5  10 M (K ) D D1 WT 9 ing to MSA as it does to mIgGs. smFcRn bound both MSA and 450.0  65.0  10 M (K ). This is in accordance with D2 and HSA, although lower binding responses were obtained values obtained by others with immobilized murine receptor compared with shFcRn binding (Fig. 3, C and D). No detectable (19). binding was seen to either albumin variant at pH 7.4. Cross-species binding to hIgG1 generated responses clearly Determination of Binding Kinetics by SPR Analyses—The stronger than those recorded for mIgG1 (Fig. 4C) and derived expressed receptor domains are normally cell bound and kinetics gave values of 0.1  0.0  10 M (K ) and 63.2 D1 exposed to circulating or pinocytosed soluble ligands. Thus, all 4.8  10 M (K ) at pH 6.0. Thus, a 85-fold decreased K D2 D1 was found compared with that of the smFcRn-mIgG1 interaction. At pH 7.4, significant concentration- dependent and reversible binding responses were obtained (Fig. 4D), and the affinity could be calculated with a K of 10 M. These data D1 are summarized in Table 1. The kinetics of cross-species IgG bind- ing have previously been recorded by SPR with IgG immobilized on the chip (44–46). This receptor:ligand orientation estimates a lower affin- ity for the interaction than that recorded here, where the receptor is immobilized. The trends were obtained, however. To evaluate binding of MSA to WT smFcRn , dilutions of monomeric MSA were injected over the immo- bilized receptor at pH 6.0, and the representative sensorgram demon- strates reversible binding (Fig. 5A). The data fitted well to a simple first-order bimolecular interaction model applied with the BIAevalua- tion software and gave a K of 9.3 WT FIGURE 4. SPR analyses of the smFcRn interaction with hIgG1 and mIgG1. Representative sensorgrams WT 0.4  10 M (Table 2). No binding of serial dilutions of mIgG1 over immobilized smFcRn at pH 6.0 (A) and 7.4 (B), serial dilutions of hIgG1 over WT WT WT immobilized smFcRn at pH 6.0 (C) and 7.4 (D). In all experiments smFcRn was immobilized by amine of MSA to smFcRn was obtained WT coupling to 100 –200 RU. Dilutions of mIgG1 and hIgG1 were injected over an immobilized smFcRn at at pH 7.4 (Fig. 5B). 25 °C. The flow rate was 50 l/min. TABLE 1 WT Kinetics of the IgG interactions with smFcRn a b c c Analyte pH k k k k K f K f a1 d1 a2 d2 D1 1 D2 2 4 4 4 3 10 /Ms 10 /s 10 /Ms 10 /s nM %nM % mIgG1 6.0 16.1  0.6 13.7  0.4 3.8  0.4 17.1  0.5 8.5  0.5 78.9 450.0  65.0 21.1 mIgG1 7.4 ND ND ND ND ND ND ND ND hIgG1 6.0 41.3  0.6 0.5  0.0 7.4  0.2 4.6  0.4 0.1  0.0 82.4 63.2  4.8 17.6 hIgG1 7.4 4.1  0.3 478.5  33.2 1.6  0.1 2.7  0.3 1169.0  0.7 84.4 168.7  30.0 15.6 a WT Dilutions of mIgG1 and hIgG1 were injected over immobilized smFcRn as shown in Fig. 4. The binding measurements were performed at pH 6.0 or 7.4. Fractional occupancies, f and f , of the two independent, parallel interactions. 1 2 The kinetic rate constants were obtained using the heterogeneous ligand binding model, which gave the best global fit using the BIAevaluation 4.1 software. The model assumes two independent, parallel reactions with immobilized smFcRn-GST. The kinetic values represent the average of triplicates. ND, not determined due to no or very low binding responses. 4830 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 7 •FEBRUARY 12, 2010 Cross-species Binding to FcRn WT WT Monomeric HSA bound smFcRn , but very weakly and shFcRn interacted with MSA at pH 6.0 (Fig. 5D) and not at with fast kinetics at pH 6.0 (Fig. 5C), while no binding at pH 7.4 pH 7.4 (supplemental Fig. 3B). The estimated K at pH 6.0 was was observed (supplemental Fig. 3A). Injection of higher con- 0.8 0.2 10 M. The kinetic measurements are summarized centrations of HSA increased binding responses, but aggrega- in Table 2. When comparing kinetics, the dissociation rates tion of HSA obscured the results. However, these data could be were found to differ dramatically and increased in the following fitted to a steady-state binding model and gave rise to an esti- order: smFcRn:MSA  shFcRn:HSA  shFcRn:MSA. No data mated K of 86.2 4.1 10 M. The results are not affected by could be obtained for the smFcRn:HSA pair, due to fast kinetics. WT the chimeric composition of smFcRn, because the same weak Taken together, shFcRn bound more strongly than WT binding responses were obtained using a fully murine form of smFcRn to both albumin species, and MSA bound more FcRn (supplemental Fig. 2). strongly than HSA to both receptor variants. Thus, an affinity hierarchy appears as follows; shFcRn:MSA  shFcRn:HSA smFcRn:MSA  smFcRn:HSA. Cross-species Competitive Bind- ing—To investigate the functional impact of cross-species binding, a constant amount of each receptor was preincubated with titrated amounts of MSA or HSA and injected over immobilized HSA or MSA. The percent inhibition of FcRn binding was calculated in each case. MSA preincubated with WT shFcRn inhibited receptor bind- ing to immobilized HSA more effi- ciently than HSA, because 3-fold more HSA than MSA was required to reach 50% inhibition (0.16 versus 0.05 M) (Fig. 6A). Furthermore, HSA was shown to inhibit WT smFcRn binding to MSA rather poorly, as in this case 10-fold more HSA than MSA was required to reach 50% inhibition (Fig. 6B). Mapping the Differences in Albu- min Binding Properties—Previ- ously, we reported that the con- served His-166, located to the WT WT FIGURE 5. SPR analyses of the shFcRn and smFcRn interaction with HSA and MSA. Representative WT 2-domain of human heavy chain, sensorgrams of serial dilutions of MSA injected over immobilized smFcRn at pH 6.0 (A) and 7.4 (B), serial WT dilutions of HSA injected over immobilized smFcRn at pH 6.0 (C), and serial dilutions of MSA injected to is crucial for binding to HSA, WT WT WT immobilized shFcRn at pH 6.0 (D). In all experiments shFcRn and smFcRn were immobilized by amine because mutation of this residue coupling to500 – 800 RU. Dilutions of MSA and HSA were injected over an immobilized receptor at 25 °C. The flow rate was 50 l/min. to alanine completely eliminate TABLE 2 Kinetics of the albumin interactions with FcRn variants a b Albumin species FcRn species FcRn variant k k K K Req a d D D 3 3 10 /Ms 10 /s M M c d MSA Mouse WT 4.2  0.5 39.4  3.1 9.3  0.4 ND MSA Human WT 3.8  0.0 3.1  0.1 0.8  0.2 ND b e HSA Mouse WT NA NA NA 86.2  4.1 HSA Human WT 2.7  1.3 12.2  5.9 4.5  0.1 4.6  0.5 HSA Mouse L166R/G167E NA NA NA 26.8  0.1 MSA Human R164L/E165G 0.7  0.1 3.4  0.1 4.8  0.1 ND MSA Mouse L166R/G167E 2.7  0.2 18.5  0.5 6.8  1.8 ND HSA Human R164L/E165G 3.2  0.1 26.3  0.2 8.2  0.1 ND Dilutions of MSA and HSA were injected over immobilized receptor as shown in Figs. 5 and 8. The steady-state affinity constant was obtained using an equilibrium (Req) binding model supplied by the BIAevaluation 4.1 software. The kinetic values represent the average of triplicates. The kinetic rate constants were obtained using a simple first-order (1:1) bimolecular interaction model. ND, not determined. NA, not acquired because of fast kinetics. The kinetic values have been published in Ref. 10. FEBRUARY 12, 2010• VOLUME 285 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4831 Cross-species Binding to FcRn residues in ligand binding, Arg-164 and Glu-165 were mutated to leucine and glycine in shFcRn, whereas Leu-166 and Gly-167 were mutated to arginine and glutamic acid in smFcRn. Titrated amounts of monomeric HSA and MSA were again injected over immobilized receptor variants at pH 6.0. Rep- resentative sensorgrams demonstrate reversible binding responses at acidic pH, and the calculated binding kinetic val- ues differ from that of the wild types (Fig. 8, A–D, and Table 2). L166R/G167E The humanized smFcRn variant bound HSA 3-fold more strongly than the wild-type mouse form, and the binding affinity for MSA was also slightly increased. Further- R164L/E165G more, rodentized shFcRn bound HSA with an affin- ity of 2-fold weaker and MSA with a 6-fold weaker affinity WT than shFcRn . Thus, exchange of human-mouse amino acids in the vicinity of the key histidine residue decreased the differ- ences in albumin-binding properties at acidic pH. However, the R164L/E165G affinity of rodentized shFcRn was not completely WT reduced to that of smFcRn , and the affinity of humanized L166R/G167E smFcRn did not totally reach the binding affinity of WT shFcRn . Also, the impact of the mutations on hIgG1 binding was investigated by ELISA. No differences in binding were detected H166A R164L/E165G for shFcRn and shFcRn compared with WT shFcRn (Fig. 8E). Mutation of two conserved glutamic acids, Glu-115 and Glu-116 (highlighted in Fig. 7B), to alanines E115A/E116A (shFcRn ), completely eliminated binding to hIgG1 (Fig. 8E). This result supports a key role for these negatively charged residues in IgG binding, as previously shown by others H168A L166R/G167E (30, 47). Both smFcRn and smFcRn bound hIgG1 like the wild-type receptor (Fig. 8F). Thus, mutation of amino acids close to the conserved histidine did not influence binding to hIgG1. Bifunctional FcRn Ligand Binding—shFcRn has been shown FIGURE 6. Competitive FcRn-albumin binding across species. A, serial dilu- to bind both hIgG and HSA simultaneously in a pH-depen- tions of HSA (0.5– 0.05 M) and MSA (0.5– 0.05 M) were preincubated with WT shFcRn (0.05 M) and injected over immobilized HSA (2600 RU). B, serial dent manner (11). Cross-species binding of both ligands to dilutions of HSA (10.0 – 0.1M) and MSA (1.0 – 0.1M) were preincubated with FcRn may reveal how they are transported and protected WT smFcRn (0.10 M) and injected over immobilized MSA (2000 RU). The from degradation in WT and transgenic mouse strains. We representative binding data are presented as percent inhibition of the FcRn binding to immobilized albumin. Injections were performed at 25 °C, and the investigated the effect of each ligand on the binding of the flow rate was 50 l/min. other by injecting IgG and albumin, from both species, sep- arately or together as a preincubated sample, over surfaces binding to HSA at acidic pH, whereas binding to hIgG is immobilized with smFcRn or shFcRn at acidic pH. Fig. 9 (A retained (10). This residue is conserved in all known FcRn and B) shows the resulting responses for binding to shFcRn sequences, including the mouse and rat HCs (Fig. 7A) (10). and smFcRn, respectively. Both receptors bound their native Mutation to alanine of the corresponding residue in the mouse ligands in an independent and additive manner. shFcRn counterpart (His-168) completely eliminated binding to HSA ignored binding to mIgG, and smFcRn bound very weakly to (100 M), and only weak binding was detected for MSA (20 M) HSA. In all cases, however, for both receptors, neither ligand when injected over a high density surface with immobilized (IgG or albumin from both species) interfered with binding mutant at acidic pH (supplemental Fig. 4, A and B). of the other. We speculated whether species differences in binding kinet- DISCUSSION ics may be caused by non-conserved amino acids found in prox- imity to His-166 in the folded molecule. Interestingly, inspec- Proper folding and cellular transport of the FcRn HC is abso- tion of the flanking amino acids revealed major non-conserved lutely dependent on association with 2m in the endoplasmic variations, because the neighboring exposed basic Arg-164 and reticulum (48). Thus, both polypeptides need to be present for acidic Glu-165 in humans are replaced by the hydrophobic generation and secretion of cell bound as well as truncated leucine and glycine residues in rodents, respectively (Fig. 7, A forms of heterodimeric FcRn. Expression of functional chi- and B). His-166 and the non-conserved amino acid residues meric FcRn has earlier been demonstrated in vivo in mice trans- (Arg-164 and Glu-165) are highlighted in the human crystal genic for the hFcRn HC (4) or the bovine FcRn HC (49). In both structure shown in Fig. 7B. To explore the putative role of these cases, the HC associates with mouse 2m into a functional 4832 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 7 •FEBRUARY 12, 2010 Cross-species Binding to FcRn injected the receptor (45, 46, 50). In this situation, the actual affinities that were calculated were lower than those obtained here using the hetero- geneous ligand binding model. However, the binding hierarchies WT were the same, with shFcRn WT ignoring mIgG and smFcRn binding better to hIgG than to mIgG. The high affinity is in agree- ment with binding studies of FcRn expressed on cells (51). Importantly, with IgG immobi- lized, the interaction between WT smFcRn and hIgG at physiologi- cal pH was barely detectable. By immobilizing the receptor, we were able to obtain kinetic data that sug- gest a difference in K of four logs D1 for the interactions at acidic and physiological pH. The higher affinity of hIgG1 for WT smFcRn compared with the smFcRn-mIgG interaction at pH 6.0 may indicate that half-life in WT mice could be overestimated. How- ever, the fact that hIgG1 also binds with reasonable affinity at physio- logical pH could counteract the effect, because it has been shown that such interaction lowers the FIGURE 7. The crystal structure of shFcRn. A, amino acids flanking His-166 (hFcRn) and His-168 (mFcRn) half-life (52). In any case, half-life located within the heavy chain 2-domain are shown. His-166 and His-168 are shown in bold. The non-con- estimations of mIgG and hIgG in served Arg-164 and Glu-165 of hFcRn, and Leu-167 and Gly-168 of mFcRn are shown in italic. B, the crystal structure of shFcRn shown in two orientations. The localization of amino acids essential for IgG (Glu-115 and WT mice show approximately the Glu-116) and albumin (His-166) binding are highlighted as blue and red spherical balls. The non-conserved same values (17). Arg-164 and Glu-165 (human) are highlighted with yellow and gray spherical balls, respectively. The FcRn heavy chains are shown in green and the 2m in orange. The figures were designed using PyMOL (DeLano Scientific) SPR analyses showed that WT with the crystallographic data of shFcRn (37). smFcRn interacted pH depen- dently with MSA with an estimated transmembrane-anchored chimeric FcRn. However, direct K of 9.3  0.4  10 M at acid pH. This is the first report on interaction studies of soluble forms of chimeric FcRn molecules in vitro kinetics of the smFcRn-MSA interaction, a finding that with ligands have not been reported. supports the role of FcRn in albumin half-life regulation in mice In this study, we show that a truncated mFcRn HC assembles (3, 53). with h2m, and that the heterodimer is secreted from HEK The remarkably long half-life of albumin was well recognized 293E cells. The functional integrity of the chimeric smFcRn was before its relationship with FcRn was discovered and utilized to extensively investigated by ELISA analyses and revealed bind- enhance the in vivo effect of short-lived therapeutic substances. ing to mIgG1, mIgG2b, hIgG1 and no detectable binding to a For instance, HSA-fused interferon 2b is now undergoing Phase H435A hIgG1 mutant. The chimeric receptor performed as a 3 trials (54), and other HSA fusions are under study. Importantly, completely murine receptor and was then utilized in a series of such constructs require animal models for preclinical evaluation. ELISA and SPR experiments to obtain new information about Recent reports have addressed the in vivo half-life of HSA cross-species ligand binding to FcRn. fused or targeted molecules in mice (25, 28) and argued that the The amino acid sequences of bovine, rat, and mouse 2m increase in half-life observed is a consequence of FcRn-medi- showed 73%, 68, and 66% homology with the human counterpart, ated rescue. Improved tumor imaging in rodents has been and the corresponding values for the FcRn HC were 76%, 64 and obtained using antitumor antigen antibody fragments geneti- 66%, respectively (supplemental Table 2 and 3). Thus, FcRn HC cally fused to HSA- or HSA-binding proteins (26, 29, 55). How- from other species may well be co-expressed with h2m in HEK ever, no complementary and comparative studies of such 293E cells to produce a variety of chimeric FcRn variants. mFcRn cross-species binding to HSA have been reported. Here, To perform SPR, the receptors were immobilized on the chip, we demonstrate a large difference in the kinetics of albumin WT and the ligands were injected. Others have immobilized IgG and binding to the mouse and human forms of FcRn. smFcRn FEBRUARY 12, 2010• VOLUME 285 • NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4833 Cross-species Binding to FcRn ther molecule shows more than half the serum persistence of endoge- nous rat albumin (56). Notably, mouse and rat FcRn HCs showed high homology (89%) (supplemen- tal Table 3), as did rat and mouse albumin sequences (90%). Thus, the rat FcRn-HSA interaction is likely as weak as the smFcRn-HSA interac- tion. Albumin-targeted molecules have been described that achieve the same half-life as endogenous albumin. This is the case with human domain antibodies selected to bind albumin (57). Two anti-rat albumin domain antibodies with low (1 M) and high (13 nM) affinity showed half-lives in rats of 43 and 53 h, respectively. Rat albumin has a half-life of 53 h, similar to the high affinity domain antibody. WT Although shFcRn ignores mIgG, it interacts strongly with MSA. The affinity for MSA was 100- fold stronger than that of the murine receptor. Based on this, one would predict that the mouse strain transgenic for the hFcRn HC would bind strongly to endog- enous MSA and protect it from degradation. This was indeed the case, and a 46% increase of the MSA levels in such mice has been observed (3). The presence of MSA bound to the human receptor, and also high serum concentrations of MSA, will surely affect rescue of HSA-associ- ated molecules that compete for the same binding site on the receptor. FIGURE 8. SPR analyses of albumin binding to rodentized and humanized FcRn variants. Representative R164L/E165G sensorgrams of serial dilutions of HSA (A) and MSA (B) injected over immobilized rodentized shFcRn Notably, the off rate of MSA is 10 L166R/G167E at pH 6.0. Serial dilutions of MSA (C) and HSA (D) injected over smFcRn . In all experiments the receptor times lower than that of HSA. variants were immobilized by amine coupling to 1000–2000 RU. Dilutions of MSA and HSA were injected over WT H166A immobilized receptors at 25 °C. The flow rate was 50 l/min. E, binding of shFcRn , shFcRn , Transgenic mice, fortified with R164L/E165G E115A/E116A WT H166A shFcRn , and shFcRn to hIgG1 at pH 6.0 in ELISA. F, binding of smFcRn , smFcRn , and serum hIgG, are useful when evalu- L166R/G167E smFcRn to hIgG1 at pH 6.0 in ELISA. The numbers given represent the mean of triplicates. ating serum persistence of engi- binds MSA with a K of 10 M. The affinity for the endoge- neered hIgGs, but the half-life of nous ligand is 10-fold higher than that for HSA, a fact that HSA variants and conjugates may well be underestimated. The WT nicely correlates with the inhibition data where smFcRn was latter is supported by the competitive data presented here WT shown to prefer MSA over HSA. This must necessarily affect where MSA efficiently inhibited shFcRn binding to HSA. the in vivo half-life of both HSA and HSA fused molecules in The binding sites for hIgG and HSA are distally localized mice in the presence of high amounts of circulating endoge- in the 2-domain of the hFcRn HC (10). In line with this, we nous albumin. When HSA-fused molecules show a moderate show that all combinations of ligands bound additively to WT increase in half-life in rodents, and not an extended half-life both receptor forms, and that shFcRn ignores mIgG while similar to that of endogenous albumin, it may simply be an binding strongly to MSA. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Feb 12, 2010

Keywords: Antibodies; Protein/Ligand Binding; Receptors/Recycling; Blood; Protein Structure; Albumin; Biodistribution; Half-life; Immunoglobulin G; Neonatal Fc Receptor

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