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Volume 270, Number 8, Issue of February 24, 1995 pp. 3958-3964
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interferon Receptor Extracellular Domain Expressed as IgG Fusion Protein in Chinese Hamster Ovary Cells
PURIFICATION, BIOCHEMICAL CHARACTERIZATION, AND STOICHIOMETRY OF BINDING (*)

(Received for publication, July 19, 1994; and in revised form, September 28, 1994)

Michael Fountoulakis (1), (§),  Cecilia Mesa (1),  Georg Schmid (2),  Reiner Gentz (¶),  ,  Michael Manneberg (1),  Martin Zulauf (3),  Zlatko Dembic (1),  Gianni Garotta (1)

From the  (1)From F. Hoffmann-La Roche Ltd., Pharmaceutical Research, Department of Gene Technology, (2)Department of Biotechnology, and (3)Department of Physics, CH-4002 Basel, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Agents that antagonize the functions of interferon (IFN) are potential pharmaceuticals against several immunological and inflammatory disorders. IFN receptor-immunoglobulin G fusion proteins (IFNR-IgG) function as antagonists of endogenous IFN and have longer half-lives in vivo in comparison with soluble IFN receptors (sIFNR), consisting of the extracellular region of the native sequence. A fusion protein comprising the extracellular domain of the human IFN receptor and the hinge, CH(2) and CH(3) domains of the human IgG3 constant region, was expressed in Chinese hamster ovary cells. The IFNR-IgG3 fusion protein was secreted into the culture medium as a 175-kDa glycoprotein and was purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion chromatography. IFNR-IgG3 bound IFN in solid phase assays and ligand blots, competed for the binding of radiolabeled IFN to the cell surface receptor of Raji cells, and inhibited the IFN-mediated antiviral activity with an efficiency at least one order of magnitude higher than that of the soluble receptor produced in the same expression system. Two IFNR-IgG3 fusion proteins bound two IFN dimers forming a complex of approximately 380 kDa. In immunodiffusion assays, the IFNR-IgG3 fusion protein did not precipitate IFN. Dissociation of bound IFN from IFNR-IgG3 was 2-fold slower than from the sIFNR produced in insect cells.

INTRODUCTION

Interferon (IFN) (^1)is a cytokine, produced by activated T lymphocytes and natural killer cells, that exerts complex functions in the control and modulation of nearly all phases of immunological and inflammatory responses(1, 2, 3) . The active protein is a homodimer with a predominant alpha-helical structure, the two subunits showing an anti-parallel organization(4) . IFN exerts its functions by interacting with a ubiquitous, specific cell receptor(5, 6, 7) , a 90-kDa glycoprotein(8, 9, 10) . In addition to IFN and IFNR, accessory proteins are required for signal transduction (11, 12, 13, 14, 15) . Recently the cloning of such an accessory factor or IFNR beta-chain was reported(16, 17) . It is not clear at present whether the accessory protein participates in ligand binding. The IFNR does not possess intrinsic tyrosine kinase activity, suggesting receptor association with specific kinases following ligand binding(18, 19, 20) . The signal transduction pathway of IFN involves phosphorylation of p91, a subunit of the IFN-stimulated gene factor-3(21, 22) .

In certain immunological disorders, IFN acts as a disease-promoting agent, and substances that antagonize its functions are potential pharmaceuticals(23, 24) . In order to obtain IFN antagonists, we engineered soluble forms of the IFN receptor (sIFNR), comprising the extracellular domain of the native protein and retaining full capacity for binding IFN(25, 26) . When administered to animals, the sIFNRs inhibited the activity of IFN, but they showed relatively short half-lives of 1-3 h in the blood and 6 h in lymphoid organs(28, 29) . Fusion proteins comprising the extracellular domain of the mouse IFN receptor and sequences of the mouse constant IgG region were active in vivo and showed an increased blood persistency (30, 31) . Because administered human sIFNR is expected to have a short half-life, we expressed in Chinese hamster ovary (CHO) cells a fusion protein comprising the extracellular domain of the human IFNR and parts of the human IgG constant chain. Here we report on the purification and characterization of this human fusion protein and the stoichiometry of its interaction with IFN.

EXPERIMENTAL PROCEDURES

Materials

Reagents for the preparation of SDS-polyacrylamide gels and protein size markers were from Bio-Rad. ^14C-Protein markers and iodinated sheep anti-mouse Ig were from Amersham. Protein G- and DEAE-Sepharose were from Pharmacia Biotech Inc. Proteolytic enzymes were purchased from Boehringer Mannheim.

IFN

Human IFN was purified from Escherichia coli according to Döbeli et al.(32) and was iodinated utilizing the chloramine-T method (33) to 2 times 10^5 cpm/ng of protein. The monoclonal antibody 69 was raised against recombinant IFN.

Soluble Human IFN Receptors Produced in CHO and Insect Sf9 Cells

The proteins were purified essentially like the soluble mouse IFNR produced in insect cells(26, 34) . The monoclonal antibody R99 was raised against the native IFNR(10) , and the polyclonal 3891 was raised against the sIFNR produced in E. coli(25) .

Analytical Methods

The fusion protein and the soluble receptors were resolved on 7.5 and 12% SDS-polyacrylamide gels, respectively, and revealed by staining with Coomassie Blue. The purity of the proteins was estimated by densitometric analysis of the stained SDS gels. If not otherwise indicated, no reducing agent was present in the sample buffer. Where it is mentioned, the proteins were reduced by addition of 10% beta-mercaptoethanol and heating at 95 °C for 5 min. Controlled reduction was performed in 5 mM dithiothreitol at room temperature for 30 min. The reaction was stopped by addition of iodoacetamide to 0.1 M final concentration. Analysis on native gels was performed as previously reported(35) . The protein concentration was determined by amino acid analysis(36) . Immunodiffusion assays were performed on 1% agarose according to Ouchterlony(37) .

Construction of Plasmids and Selection of Transfectant Cell Lines

Plasmid RCI 28.1-3A encoding the complete human IFN receptor (8) served as template for PCR. The amplified fragment contained DNA coding for the signal sequence and the extracellular domain of the receptor protein. The PCR was performed using Taq polymerase as described by the manufacturer (Perkin-Elmer). The pCD4-Hg3 (38) was used as the source of the immunoglobulin gene part. The open reading frame of the extracellular domain of the receptor cDNA was joined with that of the IgG3 gene hinge region. The human IFN receptor fragment-human IgG3 chimeric gene construct was introduced into the eukaryotic expression vector pN316 containing Rous sarcoma virus long terminal repeat promoter element and the polylinker region allowing integration of the genes of interest. (^2)Downstream from the polylinker site, pN316 included the 3`-intron and the polyadenylation site of the rat preproinsulin gene, pSV40 enhancer, the mouse dhfr gene, and the ampicillin resistance gene. CHO cells were transfected with the expression vector, and stable clones were selected as described(27) .

Fermentation of CHO Cells

Inoculum cells were cultivated in roller bottles using a mixture of Dulbecco's, Ham's F12, and Iscove's powdered media (25:25:50) with 3% fetal bovine serum (FBS). Additional medium supplements included insulin, transferrin, Pluronic F68, and Primatone RL, an enzymatic meat hydrolysate (Sheffield Products). Batch fermentations were performed in stirred tank and airlift bioreactors of up to 100 liters working volume. Cell-free supernatants were harvested by continuous centrifugation (Varifuge 20RS, Heraeus, Germany). Protease inhibitors, phenylmethylsulfonyl fluoride and benzamidine hydrochloride, were added to the culture supernatants after harvesting and to all buffers (except for the buffer used in the last purification step) to a final concentration of 1 mM and 10 mM, respectively. The culture supernatant was concentrated 10-fold by ultrafiltration using an Amicon SP20 ultrafiltration system.

Purification of the IFNR-IgG3 Fusion Protein

The concentrated supernatant was mixed with 50 ml of Protein G-Sepharose equilibrated with 15 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl (PBS) and stirred gently at 4 °C overnight. The protein solution was separated on a column from the gel, which was washed with five column volumes each of PBS and 0.1 M glycine-HCl, pH 5.0. The fusion protein was eluted with 0.1 M glycine-HCl, pH 3.0 and was neutralized with 1 M Tris-HCl, pH 8.0. The eluate was dialyzed against 20 mM sodium acetate, pH 6.0, and loaded onto a 25-ml DEAE-Sepharose column (2.5 times 5.0 cm) equilibrated with the same buffer. The ion exchanger was washed with 10 volumes of 50 mM sodium acetate, pH 4.0, and the fusion protein was eluted with 0.1 M glycine-HCl, pH 3.0. The eluate was concentrated by ultrafiltration and loaded onto a Superose-12 column HR 10/30 (Pharmacia) developed with PBS at 0.5 ml/min. One-ml fractions were collected and analyzed by SDS- and native-PAGE before pooling.

Ligand Binding and Antiviral Assays

The solid phase IFN binding assays, the ligand blots, the competition of binding of radiolabeled IFN to the cell surface receptor of Raji cells assays, and the inhibition of the IFN-mediated antiviral activity assays were performed as reported(10, 27) .

Dissociation of IFN from sIFNR and IFNR-IgG3

The kinetics of IFN dissociation from sIFNR, IFNR-IgG3, or anti-IFN monoclonal antibody 69 were performed according to Evans et al.(39) with minor modifications. In brief, 20 µg of sIFNR (carrying one binding site), 40 µg of IFNR-IgG3 (two binding sites), or 40 µg of 69 (two binding sites) were incubated with 1 µg of IFN containing 200 ng of I-IFN (3 times 10^6 cpm) in PBS in ice for 20 min. Unbound I-IFN was separated on a Superose-12 column developed with PBS. The I-IFN complexes in 750 µl were treated with 15 µg of unlabeled IFN at room temperature. At different times, 100-µl aliquots of complexes between IFN and IFNR-IgG3 or 69 were withdrawn and were added to 50 µl of Protein G-Sepharose beads, whereas complexes between IFN and sIFNR were added to 50 µl of Protein G-Sepharose beads saturated with anti-IFNR polyclonal antibody 3891, in PBS containing 2% fetal calf serum. After incubation for 4 min with gentle agitation, the beads were separated by filtering the sample through a 0.22-µm filter (Millipore Corp.). The radioactivity of the filtrate and of the beads was measured in a -counter. The time required for quantitative precipitation of the complexes (4 min) was added to the overall incubation time.

Amino Acid Analysis

Amino acid composition analysis was performed according to a modified method of Spackman et al.(40) .

Analytical Ultracentrifugation

Analytical ultracentrifugation was performed as described previously(41) .

Thermal Treatment

Soluble IFN receptor produced in insect cells (12 µg) and fusion protein (6 µg) in 1.2 ml of PBS were kept at 37 °C or were heated at 95 °C. At various times, 100-µl samples were withdrawn and kept in ice until they were analyzed for residual ligand binding capacity by ligand blots.

Proteolytic Digestion

Digestion of 1 µg of protein substrate in 1 ml of 0.1 M Tris-HCl, pH 7.4, in the presence or in the absence of 8 M urea, was performed as described(42) .

RESULTS AND DISCUSSION

Expression of IFNR-IgG Fusion Proteins

Two fusion proteins were constructed by fusing cDNA sequences encoding the extracellular domain of the human IFN receptor to sequences encoding parts of the human IgG 1 or IgG 3 heavy chains, including the hinge and constant regions CH2 and CH3. The construction of the eukaryotic expression vectors will be described elsewhere.^2 Transfected CHO cells were grown in the presence of 2-5% FBS, and the fermentation conditions were not optimized. Because the fusion proteins include cysteine residues of the hinge and constant domains of the IgG1 and IgG3 chains, they should have been produced as covalently linked homodimers. However, this happened only with the IFNR-IgG3 fusion protein, whereas IFNR-IgG1 was produced as a mixture of single chain and covalently linked homodimer in a ratio of approximately 2:1. The reasons for this discrepancy are under investigation. The purification and characterization of the IFNR-IgG1 fusion protein was not followed further in this study.

Purification Scheme

IFNR-IgG3 fusion protein was secreted into the culture medium, which was separated from the cells by centrifugation and concentrated by ultrafiltration. IFNR-IgG3 was purified in three chromatographic steps, comprising (i) Protein G-Sepharose, (ii) DEAE-Sepharose, and (iii) size exclusion chromatography. Protein G is a Type III Fc receptor that binds to the Fc region of IgG. For binding to Protein G, both chains of the fusion protein are required. IFNR-IgG3 recovered from Protein G-Sepharose was contaminated with bovine IgG, which was present in the FBS used for cell culture (Fig. 1A, lanes 3 and 8). When the FBS was passed through a Protein G-Sepharose column before fermentation, the amount of bovine IgG co-eluted with the human fusion protein was significantly reduced, but it was not completely eliminated (not shown). On nonreducing SDS-PAGE, the fusion protein migrated at approximately 175 kDa. Under the same conditions, bovine IgG co-migrated with IFNR-IgG3, so that a discrimination between these proteins was not possible (Fig. 1A, lanes 3-5). Under reducing conditions, the proteins could be clearly discriminated, since bovine IgG was resolved in heavy and light chains of 50 and 25 kDa, respectively (Fig. 1A, lanes 6 and 8-10; the light chain co-migrated with the front on 7.5% SDS gels and is not seen), whereas the hybrid protein gave rise to a 90-kDa band corresponding to a single fusion chain (Fig. 1A, lanes 8-10).

Figure 1: SDS-PAGE analysis of the human IFNR-IgG3 fusion protein after each purification stage (A) and after a second size exclusion chromatography step (B). The fusion protein was purified as described under ``Experimental Procedures.'' Electrophoresis was in the presence or absence of 10% beta-mercaptoethanol (as indicated) on 7.5% SDS gels stained with Coomassie Blue. A, lanes 1 and 6, bovine IgG (reference, 2 µg). In lane 6 only the heavy chain of bovine IgG is seen. In lanes 2-5 and 7-10, 10 µg of total protein were loaded. Lanes 2 and 7, supernatant of culture medium. The strong band represents BSA, which shows a shift in mobility under reducing conditions (lane 7). Lanes 3 and 8, eluate from Protein G-Sepharose. Lanes 4 and 9, eluate from DEAE-Sepharose. Lanes 5 and 10, eluate from Superose-12 H/R 10/30 column. M, high molecular mass markers. B, the eluate from the size exclusion step was concentrated by ultrafiltration and loaded a second time on the sizing column. Lanes 1 and 4, bovine IgG (reference). Lanes 2-3 and 5-6, eluate from the second Superose-12 step (lanes 2 and 5, 10 µg; lanes 3 and 6, 20 µg). M, high and low molecular mass markers.


Chromatography on DEAE-Sepharose, following the Protein G-Sepharose step, removed most of the bovine IgG, eluted at pH 4.5. The fusion protein was eluted at pH 3.5 and 3.0, and it still included small amounts of bovine IgG (Fig. 1A, lanes 4 and 9). Size exclusion chromatography, which followed the ion exchanger step, delivered two protein peaks, a and b, corresponding to proteins of apparent molecular masses 600 and 160 kDa, respectively (Fig. 2A). The protein bands of the fractions of peak a barely entered the native gel (Fig. 2B, lanes 2-4). When these fractions were analyzed on SDS-PAGE, the proteins co-migrated with the fusion protein of peak b (Fig. 2C, lanes 7-9), indicating that peak a included oligomeric forms of IFNR-IgG3 (Fig. 2C, lanes 2-4). The oligomeric forms were noncovalently linked (nonreducing SDS-PAGE is not shown). The SDS-PAGE analysis additionally revealed the presence of bovine IgG in the fractions of peak a (Fig. 2C, lanes 2-4; the 55-kDa band). Peak b comprised highly purified fusion protein (Fig. 2B, lanes 8-10, and Fig. 2C, lanes 7-9), still contaminated, however, with approximately 2% bovine IgG (Fig. 1A, lanes 5 and 10). A second size exclusion chromatography cycle of the fusion protein of peak b yielded a more than 99% pure fusion protein preparation with less than 1% bovine IgG (Fig. 1B, lanes 2-3 and 5-6). The overall recovery was approximately 60%, as judged by ligand blots. The described method did not completely remove bovine IgG. Bovine IgG was completely removed when Protein G-pretreated FBS was used for cell culture and the fusion protein was purified following the described purification scheme over Protein G-Sepharose, DEAE-Sepharose, and sizing steps (data not shown).

Figure 2: Protein elution profile (A), native (B), and SDS-PAGE analysis (C) of the proteins eluted from the sizing column. A, the proteins were loaded on a Superose-12 H/R 10/30 column developed with PBS. B, analysis of selected fractions on a 5% native gel stained with Coomassie Blue. Lane 1, starting material loaded; lanes 2-10, fractions eluted from the column. Fractions 20-22 (lanes 2-4) contained oligomeric forms of the fusion protein. *, fusion protein, homodimer. C, the proteins were analyzed under reducing conditions on a 7.5% SDS gel stained with Coomassie Blue. Lane 1, proteins loaded. The 55-kDa band represents bovine IgG (heavy chain). Lanes 2-9, fractions eluted from the sizing column. M, molecular mass markers.**, fusion protein, single chain.


Characterization of the IFNR-IgG3 Protein

Amino acid sequence analysis from the N-terminal end revealed that the protein was processed properly and that the signal peptide sequence was cleaved off. The protein was heterogeneous at the N terminus since 50% of the molecules started with Glu-Met-Gly-Thr-Ala-Asp- and the rest with Gly-Thr-Ala-Asp-. Amino acid composition analysis showed that the protein had the expected composition. A molecular mass of 114 kDa was calculated for IFNR-IgG3 (comprising the two covalently linked chains). Gel filtration and analytical ultracentrifugation revealed a molecular mass of approximately 160 kDa for the fusion protein, suggesting that it exists as monomer in physiological buffer. The difference between the apparent and calculated mass is caused by glycosylation. (^3)In immunoblots, the fusion protein was detected by specific antibodies raised against the native IFNR. In immunodiffusion assays on agarose, the fusion protein was precipitated by the polyclonal antibody R3891 but not by the monoclonal R99 (data not shown).

Stability of IFNR-IgG3

IFNR-IgG3 retained full ligand binding capacity when incubated at 37 °C for 8 days and 20% of its activity when treated at 95 °C for 1 h, as judged by ligand blots. The ligand binding capacity of the fusion protein decreased after 10 freezing-thawing cycles to approximately 10% of the original value and then remained constant for up to 30 cycles. The resistance of IFNR-IgG3 to proteolysis in vitro was similar to that of sIFNRs (42, 43) during folding and in the folded state (data not shown).

Binding of IFN

IFNR-IgG3 bound IFN on solid-phase and protein blot assays (not shown). The bivalent IFNR-IgG3 inhibited the I-IFN binding to the natural receptor of Raji cells with an IC of approximately 0.17 nM (Fig. 3A). The molecular mass of the IFNR-IgG3 was considered to be 114 kDa (glycosylation was not taken into account). Under the same conditions, unlabeled IFN also competed with an IC of 0.20 nM, whereas the monoclonal antibody 69 competed with an IC of approximately 1.2 nM (Fig. 3A). Thus, the fusion protein competed at least one order of magnitude more efficiently (IC = 0.17 nM) in comparison with the sIFNR produced in CHO cells (IC = 2.3 nM) (Fig. 3A) and the sIFNRs produced in baculovirus-infected insect cells (IC = 8 nM) (27) or in E. coli (IC = 15 nM)(25) . In inhibition of the cell cytopathic effect (Fig. 3B), IFNR-IgG3 performed approximately 20-fold more efficiently (IC = 1.20 nM) than the sIFNR from CHO cells (IC = 23 nM). In comparison, 69 inhibited with an IC of 0.05 nM (Fig. 3B).

Figure 3: Competition of binding of radiolabeled IFN to Raji cells (A) and inhibition of IFN-mediated antiviral activity (B). The assays were performed as described in (27) . A, IFNR-IgG inhibited the binding of 2 ng of I-IFN to the receptor of 10^7 Raji cells with an IC of 0.17 nM () and the sIFNR from CHO cells inhibited with an IC of 2.30 nM (bullet). circle, inhibition by unlabeled IFN; , inhibition by anti-IFN antibody 69. B, the inhibition of antiviral activity was studied on human WISH fibrostast cells infected with encephalomyocarditis virus. IFNR-IgG3 inhibited with an IC of 1.20 nM (), the CHO cell-derived sIFNR with an IC of 23 nM (bullet), and the antibody 69 with an IC of 0.05 nM (Delta). AVA, antiviral activity.


We investigated the exchange rate of IFN bound by IFNR-IgG3 and for comparative reasons by sIFNR and anti-IFN antibody 69. The kinetics of dissociation were studied by mixing radiolabeled IFN with either of the three proteins, adding the excess of unlabeled IFN, and measuring the time-dependent release of the iodinated ligand. IFN complexed with IFNR-IgG3 was released approximately 2-fold more slowly in comparison with that complexed with 69 or the sIFNR (Fig. 4). Table 1summarizes the performance of the three proteins in ligand binding. The increased retention time may be essential if IFNR-IgG3 will be used as antagonist of endogenous IFN. Similarly, the better performing in vivo tumor necrosis factor receptor p55 fusion protein had a significantly slower exchange rate for bound tumor necrosis factor alpha in comparison with the p75 fusion protein(39) .

Figure 4: Dissociation of IFN bound to the IFNR-IgG3 fusion protein. The kinetics of dissociation of complexed I-IFN (3 times 10^6 cpm) were performed as stated under ``Experimental Procedures.'' Dissociation of I-IFN bound to IFNR-IgG3 (), to sIFNR from insect cells (bullet), and to monoclonal anti-IFN antibody 69 (). The bars represent standard error of the mean of four experiments.


After reduction, IFNR-IgG3 did not show IFN binding activity on ligand blots. Controlled reduction of the 175-kDa fusion protein resulted in generation of a 90-kDa single-chain species with IFN binding capacity suggesting that the four disulfides of the extracellular domain of the IFNR, all of which are essential for ligand binding(44, 45) , are more stable than the disulfides connecting the two IgG3 heavy chains. Dialysis of the completely reduced fusion protein resulted in partial reconstitution of the biological activity of the 90-kDa single chain species only, but not of the 175-kDa IFNR-IgG3. Thus, the disulfide bonds of the IFNR domain of the fusion protein were reconstituted, whereas the disulfides between the two IgG3 constant domains were not formed (data not shown).

Stoichiometry of Binding

We investigated the stoichiometry of binding between IFNR-IgG3 and IFN by native gels, gel filtration, amino acid composition analysis, and analytical ultracentrifugation. The components were mixed taking into consideration a molecular mass of 114 kDa for the fusion protein and 32 kDa for IFN, since this cytokine exists as a dimer in physiological buffer(41) . Based on data of native gel analysis of different ligand-fusion protein ratios, IFNR-IgG3 and IFN were mixed at a ratio of 1:1, and the complex was chromatographed on a size exclusion column. An apparent molecular mass of approximately 400 kDa was found for the complex (Fig. 5A). The complex, as eluted from the sizing column, as well as the nonmixed components were subjected to amino acid composition analysis. The observed amino acid ratios of the components and of their complex were introduced into a computer program(46) , and an IFNR-IgG3 fusion protein-IFN dimer molar ratio of 1:1 was found. Analytical ultracentrifugation delivered for the complex an apparent molecular mass of 380 kDa. Taking into consideration the results of the four analytical approaches, we conclude that IFNR-IgG3 and IFN dimer interact at a molar ratio of 1:1 and that in physiological buffer they form a complex consisting of two IFNR-IgG3 molecules and two IFN dimers ((2 times 160) + (2 times 32) = 384; the glycosylated fusion protein was detected as a 160-kDa species by size exclusion chromatography and analytical ultracentrifugation). The apparent molecular mass values of the complexes determined by the different approaches are in good agreement with each other. The minor deviations may be caused by the high glycosylation grade of the fusion protein (approximately 60 kDa).

Figure 5: Size exclusion chromatography (A) and native gel analysis (B and C) of IFNR-IgG3 fusion protein-IFN dimer complexes. A, IFNR-IgG3 (440 µg) and IFN (220 µg) were mixed and chromatographed on a Superose-12 column developed with PBS at 0.4 ml/min. The positions of elution of the peaks of standard proteins (670, 158, and 44 kDa) are indicated. B, 2 µg of IFNR-IgG (f) were mixed with increasing amounts of IFNDelta10 (lanes 2-5) or IFNDelta0 (lanes 6-8) at the indicated ratios, and the complexes were analyzed on a 5% native gel stained with Coomassie Blue. One major complex (c) was formed. When IFN was added at ratios 1:3 and 1:4, additional broad bands were detected (*) (lanes 4-5 and 7-8). C, IFNDelta10 (1.3 µg) was mixed with increasing amounts of IFNR-IgG3 (f) as indicated. Analysis was as stated under B. One complex (c) was formed. In excess of IFN, additional weak bands (*) were visible (lanes 2-4). IFNR-IgG3 added in excess remained uncomplexed (f, lanes 6-8).


We further studied whether the fusion protein and IFN dimer form complexes larger than the 380-400-kDa complex detected by gel filtration and analytical ultracentrifugation. IFNR-IgG3 and IFN were mixed at different ratios, and the complexes were analyzed by nondenaturing gels (Fig. 5, B and C). Fusion protein and IFN dimer formed in all cases one major complex (c) for which amino acid analysis revealed a ratio of 1:1 (Fig. 5B, lanes 2-8 and Fig. 5C, lanes 2-8). In excess of IFN, additional bands migrating between the fusion protein f and the complex c were visible, suggesting the formation of complexes in which two IFN dimers were bound by one bivalent IFNR-IgG3 fusion protein (Fig. 5B, lanes 4-5 and 8, and Fig. 5C, lane 4; these bands are broad and not clearly seen). Similar complexes were formed by one IFN dimer bound by one sIFNR molecule when IFN was added in excess to the sIFNR, although when the sIFNR was available in adequate amounts, two soluble receptor molecules always bound one IFN dimer(41) .

When IFNR-IgG3 was added in excess, again one complex c was formed. The excess of IFNR-IgG3 remained uncomplexed (Fig. 5C, lanes 6-8). Thus, no complexes larger than complex c were detected. Formation of such complexes would suggest an agglutination-like situation in which the fusion protein and IFN could form precipitate. In immunodiffusion assays on agarose, IFNR-IgG3 did not precipitate IFN, behaving like a nonprecipitating monoclonal antibody. IFN was not precipitated by the anti-IFN monoclonal antibody 69 either (data not shown).

In previous studies, using sIFNRs produced in eukaryotic expression systems, we found that one IFN dimer is bound by two receptor molecules(35, 41) . The one human IFN dimer-two receptor relation was confirmed later by other groups(47) . Applying chemical cross-linking and using high concentrations of two cross-linkers simultaneously, they showed the generation of a 240-kDa product, likely consisting of one IFN dimer bound by two native receptor molecules, thus confirming dimerization of the native receptor in the presence of ligand.

Based on predicted homology between IFNR and members of the hematopoietic receptor family (48) and in analogy to the crystal structure of the human growth hormone receptor(49) , we proposed a model for the IFN ligand-receptor interaction(45) . According to that model, the extracellular part of the IFN receptor consists of two Ig-like domains linked in a V-shaped structure. The two domains are connected with one essential disulfide (Cys-Cys). The region at the convergence of the two Ig-like domains most likely includes the ligand binding domain of the receptor. That this region is essential for ligand binding was confirmed by studies with mutant IFN receptors carrying domains of both the human and mouse species(50) .

In this study, applying four approaches, we found that two IFNR-IgG3 molecules, carrying two ligand binding sites each, bind two IFN dimers. Such a complex could be formed if each IFN dimer interacts with one of the IFN binding regions of one fusion protein and one of the binding regions of a second fusion protein (Fig. 6A). The model of the IFNR-IgG3 fusion protein-IFN dimer binding (Fig. 6A) shows that the stoichiometry of interaction is responsible for the higher ligand binding affinity of the fusion protein and for the better performance in competition of binding and inhibition of antiviral activity, in comparison with soluble receptors, in which case two receptor molecules bind only one ligand dimer (Fig. 6B).

Figure 6: Schematic representation of the interaction between IFNR-IgG3 fusion protein and IFN (A) and sIFNR and IFN (B). A, two IFNR-IgG3 fusion protein molecules bind two IFN dimers, forming a complex of approximately 380 kDa. For such a complex to be formed, each IFN dimer should be bound by one of the ligand binding domains of either of the two bivalent fusion protein molecules. B, two sIFNR molecules bind one IFN dimer ( (35) and (41) ). A and B, the two Ig-like domains of the IFNR part of each fusion protein (A) or soluble receptor (B) are shown in black. The IgG3 part is shown in gray (A). The connecting line between the IgG3 domains represents disulfides. The two subunits of the IFN dimer are shown in white (A and B).


Conclusion

We produced in CHO cells a human IFNR-IgG3 fusion protein comprising the extracellular domain of the IFN receptor and parts of the human IgG 3 heavy chain and worked out a purification method that delivered homogeneous IFNR-IgG3. We investigated the stoichiometry of its interaction with the ligand, finding that two molecules of IFNR-IgG3 bind two IFN dimers and explaining its superior performance in comparison with soluble receptors. The fusion protein has a slower IFN-exchange rate and is expected to have a longer half-life in vivo in comparison with the sIFNRs; therefore, it may prove useful as an antagonist of endogenous IFN in the treatment of immunological and inflammatory diseases.

FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: F. Hoffmann-La Roche Ltd., PRPG, Bldg. 15-16, CH-4002 Basel, Switzerland.

Present address: Human Genome Sciences Inc., Rockville, MD.

(^1)
The abbreviations used are: IFN, interferon ; CHO, Chinese hamster ovary; FBS, fetal bovine serum; IgG3, immunoglobulin G heavy chain 3; sIFNR, soluble interferon receptor; IFNR-IgG3, interferon receptor-immunoglobulin G3 fusion protein; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.

(^2)
C. Kürschner, N. Knezevic, G. Garotta, and Z. Dembic, manuscript in preparation.

(^3)
Mesa, C., Dembic, Z., Garotta, G., and Fountoulakis, M.(1995) J. Interferon Res., in press.

ACKNOWLEDGEMENTS

We thank M.-C. Boy, J.-F. Juranville, K. DiPadova, and N. Wild for technical assistance, A. Friedlein for sequence analysis, and Dr. H. Lötscher for critical reading of the manuscript.

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