Construction and Binding Kinetics of a Soluble Granulocyte-Macrophage Colony-stimulating Factor Receptor alpha -Chain-Fc Fusion Protein*

Cristina Monfardini, Mohana Ramamoorthy, Helga Rosenbaum, Qiong Fang, Paul A. Godillot, Gabriela Canziani, Irwin M. ChaikenDagger , and William V. Williams§

From the Department of Medicine, Rheumatology Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an alpha -chain (GM-CSFRalpha ), which specifically binds GM-CSF, and a beta -chain (beta c), shared with the interleukin-3 and interleukin-5 receptors. GM-CSFRalpha exists in both a transmembrane (tmGM-CSFRalpha ) and a soluble form (sGM-CSFRalpha ). We designed an sGM-CSFRalpha -Fc fusion protein to study GM-CSF interactions with the GM-CSFRalpha . The construct was prepared by fusing the coding region of the sGM-CSFRalpha with the CH2-CH3 regions of murine IgG2a. Purified sGM-CSFRalpha -Fc ran as a monomer of 60 kDa on reducing SDS-polyacrylamide gel electrophoresis but formed a trimer of 160-200 kDa under nonreducing conditions. The sGM-CSFRalpha -Fc bound specifically to GM-CSF as demonstrated by standard and competitive immunoassays, as well as by radioligand assay with 125I-GM-CSF. The sGM-CSFRalpha -Fc also inhibited GM-CSF-dependent cell growth and therein is a functional antagonist. Kinetics of sGM-CSFRalpha -Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems. In the first, the sGM-CSFRalpha -Fc was bound to immobilized staphylococcal protein A on the biosensor surface, and binding kinetics of GM-CSF in solution were determined. This revealed a rapid koff of 2.43 × 10-2/s. A second set of experiments was performed with GM-CSF immobilized to the sensor surface and the sGM-CSFRalpha -Fc in solution. The dissociation rate constant (koff) for the sGM-CSFRalpha -Fc trimer from GM-CSF was 1.57 × 10-3/s, attributable to the higher avidity of binding in this assay. These data indicate rapid dissociation of GM-CSF from the sGM-CSFRalpha -Fc and suggest that in vivo, sGM-CSFRalpha may need to be present in the local environment of a responsive cell to exert its antagonist activity.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Granulocyte-macrophage colony-stimulating factor (GM-CSF)1 plays an important role in myeloid differentiation and is involved in many inflammatory and immune processes. GM-CSF is a member of the four-helix bundle family of cytokines which also includes growth hormone, interleukin-2 (IL-2), IL-4, and IL-5. GM-CSF activity is mediated by specific cellular receptors (GM-CSFR), which belong to a supergene family (1-7). The GM-CSFR consists of an alpha -chain (GM-CSFRalpha ), specific for GM-CSF (4), and a beta -chain (beta c), which can also associate with IL-3 and IL-5 receptor alpha -chains (5). The GM-CSFRalpha expressed in the absence of beta c binds GM-CSF but with a lower affinity than the heterodimeric receptor (8).

Naturally occurring soluble forms of cytokine receptors, including a soluble form of GM-CSFRalpha (sGM-CSFRalpha ), have been described (9-11). sGM-CSFRalpha has been cloned from human placenta (11) and human choriocarcinoma cells (9) and has been detected in the supernatant of a human choriocarcinoma cell line (10). Previous work in our laboratory utilized polymerase chain reaction (PCR) to amplify and clone both the transmembrane and the soluble forms of the GM-CSFRalpha from a human GM-CSF-dependent myelomonocytic cell line (AML193). Supernatants from sGM-CSFRalpha -transfected cells, but not transmembrane GM-CSFRalpha (tmGM-CSFRalpha )-transfected cells, inhibited GM-CSF immunoprecipitation by neutralizing monoclonal antibody (mAb) 126.213 and inhibited GM-CSF-dependent cellular proliferation. These experiments indicated that sGM-CSFRalpha binds GM-CSF and exhibits functional antagonist activity in vitro (12).

The GM-CSF antagonist activity of sGM-CSFRalpha could play a role in the regulation of biological responses to GM-CSF. Biological effects mediated by the sGM-CSFRalpha in vivo would be dependent on its binding characteristics. For example, if the sGM-CSFRalpha binds GM-CSF with high affinity, forming a stable complex, the sGM-CSFRalpha would be able to sequester GM-CSF away from tmGM-CSFRalpha -bearing cells. In this way, sGM-CSFRalpha could act at a distance, perhaps by transporting GM-CSF away from responsive cells. In contrast, if the sGM-CSFRalpha binds with lower affinity, particularly with a rapid off-rate, sGM-CSFRalpha would need to be present in the local cellular environment where GM-CSF is present to exert its antagonist activity. Thus, the binding kinetics of the sGM-CSFRalpha will have a major impact on its in vivo functional role, and determining the kinetic properties of the protein would help determine the mechanistic nature of its biological role. Unfortunately, production and purification of sufficient sGM-CSFRalpha for binding studies have been problematic because production by cell lines is limited, and simple purification methods are not available. Therefore, we prepared a DNA-construct fusing the sGM-CSFRalpha to the Fc region of mouse IgG2a. Here we describe the cloning, production, purification, binding, and biological activity of this sGM-CSFRalpha -Fc fusion protein, with preliminary analyses of binding kinetics.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning of sGM-CSFRalpha -Fc

sGM-CSFRalpha cDNA clones were available as described previously (12). sGM-CSFRalpha cDNA and the CH2-CH3 cDNA region of mouse anti-reovirus type 3 mAb 9BG5 (IgG2a) (13) were amplified using primers containing restriction endonuclease sites for restriction enzymes BamHI and SalI for cloning the construct into the pBabe-puro vector and including sequences that allowed their use in the splicing by overlap extension (SOE) step for the preparation of the construct. sGM-CSFRalpha cDNA from clone 9 was PCR amplified using these primers (restriction sites underlined): sGM-CSFRalpha (BamHI), 5'-TGACCGGATCCATGCTTCTCCTGGTGACAA; and sGM-CSFRalpha (SOE), 3'-CCACCCAAGAGGTTAGGTGCGTTTGTCTTTGATCTGTGGAA.

The CH2-CH3 region (Fc region) of mouse anti-reovirus mAb 9BG5 was amplified from cDNA prepared from the hybridoma according to our prior protocols (12) with the following primers: Fc (SOE), 5'-TTCCACAGATCAAAGACAAACGCACCTAACCTCTTGGGTGG; Fc (SalI), 3'- ATGCCGTCGACCTATTTACCCGGAGTCCGGGA.

These primers were used with cDNAs and Taq polymerase as described previously (14, 15) to amplify the sGM-CSFRalpha and Fc. The program of amplification was 94 °C for 1.5 min followed by 25 cycles at 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, with a final cycle at 72 °C for 5 min. The PCR products were stored at 4 °C until used. A 2% analytical agarose-gel electrophoresis revealed a band of 1,100 bp and a band of 650 bp, respectively, for sGM-CSFRalpha - and CH2-CH3-amplified products, which were therefore purified on a 1% preparative low melting agarose gel (NuSieve, FMC BioProducts, Rockland, ME), extracted with phenol/chloroform (16), and used in the SOEing step.

The SOEing step program consisted of two phases. In the first phase (94 °C for 1 min followed by five cycles at 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min) the two amplified cDNA products worked as primers for each other to obtain the construct. In the second phase (94 °C for 1 min followed by 25 cycles at 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min) the primers sGM-CSFRalpha 5' and Fc 3' were added and the construct amplified. A 2% analytical agarose gel indicated a SOEing product of the desired size. After treatment of SOEing mixture with proteinase K to destroy Taq polymerase, the construct was extracted following the phenol/chloroform protocol and precipitated with ethanol. The construct and the pBabe vector (17, 18) were digested with restriction enzymes BamHI and SalI. After alkaline phosphatase treatment of pBabe purified on a 1% preparative low melting temperature agarose gel, ligation was performed following standard procedures (16, 19).

The product was transformed into Escherichia coli DH5alpha cells that were plated into LB plates; and minipreparations were made from colonies. Plasmid DNA from minipreps were digested with BamHI/SalI to identify the presence of the desired insert. After amplification of cells with the right insert, plasmid DNA was purified on a Qiagen column (Qiagen Inc., Chatsworth, CA). The purified DNA was checked using two different pairs of restriction enzymes (BamHI/SalI and BamHI/XbaI), and the expected size of the inserts was confirmed on a 2% analytical agarose gel (~350 bp and ~4,750 bp for the BamHI/SalI digestion, and ~1,200-1,300 bp, ~2,500 bp, and ~4,300 bp for the BamHI/XbaI digestion). The sequence of the insert was also confirmed by sequencing according to previously published protocols (20).

Transfection of PA317 Cells, Transduction into SP2/0 Cells

PA317 cells (a retroviral packaging line (17, 18)) were transfected with the purified DNA using LipofectAMINE reagent (Life Technologies, Inc.) and the described protocol (21). Briefly, 2 × 105 cells in 2 ml of DMEM (DMEM containing L-Gln, oxalate/pyruvate/insulin, penicillin/streptomycin, and 10% fetal calf serum) were seeded in each well of a six-well culture plate and grown until 80% confluence. 1.5 µg of DNA in 100 µl of serum-free DMEM was added to 12 µl of LipofectAMINE reagent diluted to 100 µl with serum-free DMEM. After gentle mixing and incubation at room temperature for 45 min to allow the DNA-liposome complex to form, the complex solution was diluted to 1 ml with serum-free DMEM and added to the rinsed adherent cells. Cells were kept in a 37 °C 5% CO2 incubator for 5 h and then rinsed, and serum-containing DMEM was added. 72 h after transfection, the cells were passed 1:10 into DMEM containing 5 µg/ml puromycin. Cells were grown until confluent, split twice, and grown for 1 week in medium without puromycin. The supernatant was then used to transduce SP2/0 cells.

3 × 106 SP2/0 cells, grown in RPMI medium, containing L-Gln, sodium pyruvate, penicillin/streptomycin, 25 mM Hepes, and 15% fetal calf serum, was resuspended in 2 ml of medium with 0.5 ml of PA317 supernatant and 10 µl of Polybrene (0.4 mg/ml). After 3 h of incubation, 8 ml of RPMI medium was added; after 2 days of incubation, cells were grown in medium containing puromycin. SP2/0 cells were then cloned and the presence of insert confirmed by PCR. An enzyme-linked immunosorbent assay (ELISA) (see below) was performed on PCR-positive clone supernatants to identify the clones producing the fusion protein.

Purification and Characterization of sGM-CSFRalpha -Fc

Clones that presented the highest binding to GM-CSF in two ELISAs were expanded, and the fusion protein from the supernatants was purified on a staphylococcal protein A (SPA) column. To 140 ml of filtered supernatant glycine and NaCl were added to reach, respectively, 1.5 and 3 M final concentrations; the pH was adjusted to 8.0. After loading the sample, the column was washed with 10 mM boric acid, 3 M NaCl, pH 8.9, and eluted with 0.1 M sodium citrate, pH 3.5 (fractions collected into tubes containing 1 M Tris, pH 9.5). Elution fractions containing proteins were concentrated using Centricon-30 (30 kDa cutoff) filtration systems (Amicon, Beverly, MA).

The sGM-CSFRalpha -Fc fusion protein was analyzed by SDS-polyacrylamide gel electrophoresis. An 8% acrylamide gel, 1.5 mm thick, was used in an electrophoresis system to analyze both oxidized and reduced forms of the fusion protein. 45 µg of purified sGM-CSFRalpha -Fc fusion protein purified from various cell clones was loaded into gels, run at 100 volts for ~1.5 h using 25 mM Tris, 250 mM glycine, 0.1% SDS, pH 7.5, and then stained with Coomassie Blue.

A Sephacryl S-200HR gel filtration column (Pharmacia Biotech Inc.) was packed into a C-16/70 column (215 ml of resin) to analyze 500 µl of 0.676 mg/ml fusion protein preparation from clone 25, using 50 mM Tris, 150 mM NaCl, pH 7.5, buffer, 0.8 ml/min flow rate, 1 mm/min chart speed, and collecting 1.2-ml fractions.

Western Blot Analysis

Western blot analysis was performed using mAbs against GM-CSFR. 75 µg/well of both oxidized and reduced SPA-purified sGM-CSFRalpha -Fc fusion protein were loaded into three wells of each of two 8% polyacrylamide gels. After the electrophoretic run as reported previously, the gels were cut in thirds, each strip containing two wells, one with the sample and one with the high molecular mass markers, and transferred to Immobilon-P membranes (Millipore, Bedford, MA) for 1 h at room temperature, under stirring conditions, using 50 mM Tris, 150 mM NaCl, 20% methanol, pH 7.5, as the transfer buffer. The membranes were then blocked overnight at 4 °C with 5% non-fat dry milk in 50 mM Tris, 150 mM glycine, 0.05% Tween, pH 7.5 (5% NFDM in TTBS). The next morning they were washed three times for 10 min with TTBS, and the three pairs of oxidized and reduced membrane strips were incubated for 2 h at room temperature with one of the following monoclonal antibodies diluted in TTBS: (a) 7 µg/ml mouse IgG2a anti-human GM-CSFR (Pharmingen, San Diego) (this antibody binds GM-CSFR on the same site recognized by GM-CSF); (b) 7 µg/ml mouse IgM anti-human GM-CSFR (Pharmingen) (this antibody binds GM-CSFR on a site different from that recognized by GM-CSF); (c) no first antibody. The membranes were then washed three times for 2 min with 5% NFDM in TTBS and twice for 2 min with TTBS. Samples from a and b were then incubated for 2 h at room temperature, under shaking conditions, with a 1:40,000 dilution of biotin-goat anti-mouse IgG (Fab-specific) (Sigma) in TTBS. After washing the membranes three times for 2 min with 5% NFDM in TTBS and twice for 2 min with 1 × TTBS, they were incubated for 2 h at room temperature, under shaking conditions with 0.5 µg/ml avidin-horseradish peroxidase (HRP; Pierce) in 1 × TTBS; sample c was incubated under same conditions with goat anti-mouse IgG (H+L) HRP (Life Technologies, Inc.). The membranes were washed six times with 1 × TTBS, and the TMB substrate solution was added (Kirkegaard and Perry Laboratories, Gaithersburg, MD) until color development. The color reaction was then stopped by distilled water rinsing.

ELISAs

Clone supernatants were tested in three types of ELISAs. In the first type, ELISA plates (Dynatech Laboratories, Chantilly, VA) wells were coated with 50 µl of 10 µg/ml GM-CSF (Sargamostin Leukine, Immunex Corporation, Seattle) in 0.1 M NaHCO3, and after overnight incubation at 4 °C they were washed five times with 1 × PBST and blocked with 200 µl of 2% bovine serum albumin in 1 × PBST at 37 °C for 1 h. The plates were washed five times with 1 × PBST, and 50 µl of clone supernatants was added, incubated overnight at 4 °C, and washed seven times with cold 1 × PBST. 100 µl of 1/3,000 cold dilution in 1 × PBST of goat anti-mouse IgG (H+L) HRP was added per well and the plate kept overnight at 4 °C. After seven washes with cold 1 × PBST, 100 µl of 0.1 mg/ml substrate TMB dihydrochloride (Sigma) in 0.05 M phosphate citrate, 0.03% sodium perborate buffer, pH 5.0, was added per well. After color development at room temperature for 10 min, the enzymatic reaction was stopped with 20 µl of 2 N H2SO4 per well and the plate read at 450 nm.

In the second ELISA the wells were coated with 50 µl of 5 µg/ml SPA (Sigma) in 0.1 M NaHCO3 for 1 h at 37 °C, washed five times with 1 × phosphate-buffered saline with 0.1% Tween-20 (PBST), and blocked with 200 µl of 2% bovine serum albumin in 1 × PBST at 37 °C for 1 h. The wells were washed five times with 1 × PBST, 150 µl of clone supernatants was added, and the wells were incubated overnight at 4 °C and washed seven times with cold 1 × PBST. 50 µl of 10 ng/ml biotinylated GM-CSF (developed by standard methods (22)) was added per well, and the plate was incubated overnight at 4 °C, washed seven times with cold 1 × PBST, and 100 µl of 0.1 µg/ml avidin-HRP conjugate added per well. After incubation overnight at 4 °C and seven washes with cold 1 × PBST, 100 µl of substrate was added to each well, and after color development at room temperature for 10 min, the enzymatic reaction was stopped with 20 µl of 2 N H2SO4 per well and the plate read at 450 nm.

The third was a competitive ELISA, used to test highly concentrated fusion protein preparations after purification on an SPA column. An ELISA plate was coated with SPA and blocked as reported previously. 50 µl of 1 mg/ml sGM-CSFRalpha -Fc was added per well and incubated overnight at 4 °C. After three washes with 1 × PBST, 50 µl of unmodified ("cold") GM-CSF was added at various dilutions and incubated for 1 h at 37 °C. 50 µl of biotinylated-GM-CSF at 10, 1, and 0.1 ng/ml was added per well, and the plate was incubated for another hour at 37 °C. After washing, binding was detected by avidin-HRP as reported above.

Radioligand Assay

Binding of purified sGM-CSFRalpha to 125I-GM-CSF was also tested as follows. The wells of a radioimmunoassay plate (Wallac, Gaithersburg, MD) were coated with 50 µl of 5 µg/ml SPA overnight at 4 °C, washed three times with PBST, and blocked with 200 µl of 2% bovine serum albumin in 1 × PBST at 37 °C for 2 h. The plate was washed three times with PBST and then 50 µl of purified fusion protein per well was added at 500, 250, 125, 62.5, 31, 15.5, and 0 µg/ml in 2% bovine serum albumin and PBST; as positive control anti-GM-CSF mAb 126.213 was used at same dilutions. After a 3-h incubation at 37 °C, the plate was washed three times with cold PBST and incubated with 25 µl of 388 pM 125I-GM-CSF (1 × 105 to 3 × 105 cpm) per well, with or without a saturating amount (100 nM) of cold GM-CSF at room temperature for 1 h. The plate was quenched on ice, washed three times with ice-cold PBST, and the wells were cut out from the plate, transferred into tubes, and the cpm counted.

Proliferation Assay

Inhibition of GM-CSF-dependent cells line MO7E (24 h) (from R. Zollner, Genetics Institute, Cambridge, MA) by sGM-CSFRalpha was tested as follows. 104 cells/well were incubated for 1 day with or without 250 pM GM-CSF in the presence of different concentrations of sGM-CSFRalpha (250, 50, 10, 2, and 0 µg/ml). 20 µl of 5 mg/ml MTT (Sigma) in 1 × PBS was added per well, and after 5 h 100 µl of 10% SDS in 0.1 N HCl was used to solubilize the precipitated crystals. After overnight incubation at 37 °C, A570 nm was detected. A parallel assay was set as control, using IL-2-dependent CTLL cells (5 × 103 cells/well) in the presence of 20% concanavalin A supernatant as the stimulus.

Biosensor Assay

An IAsys biosensor was used to characterize binding kinetics of sGM-CSFRalpha -Fc to GM-CSF. All experiments were done at 25 °C.

Immobilization and Regeneration Conditions

GM-CSF and SPA immobilization on a carboxymethyl-dextran cuvette (IAsys) was performed in the instrument using reagents supplied in the coupling kit as recommended by the manufacturer. Briefly, the cuvette was first washed with 200-µl washes of alternatively deionized water and 10 mM HCl to swell the cuvette matrix and finally equilibrated in PBST. The carboxymethyl-dextran layer on the sensor chip was then activated by a 10-min incubation with a mixture of 100 µl of 13 mg/ml N-hydroxysuccinimide and 100 µl of 76 mg/ml EDC to form succinimidyl esters that are reactive with amino groups. After washes with PBST, 200 µl of 10 µg/ml GM-CSF or SPA in 10 mM sodium acetate, pH 5, was added to the cuvette, and after a response plateau was reached signifying completion of the reaction, the cuvette was washed out with PBST. The remaining activated groups were blocked by injection of 200 µl of 1 M ethanolamine, pH 8.5, for 2 min. After reequilibration with PBST, the cuvette was ready for the binding experiments. The cuvette matrix was regenerated to remove bound ligand using 200 µl of either 0.5 M sodium carbonate, pH 11.0, or 10 mM HCl and 0.5 M NaCl and reequilibrated with PBST.

Binding Assays

Immobilized GM-CSF-- 200 µl of sGM-CSFRalpha -Fc at 75, 300, 600, and 900 nM in PBST was added to the cuvette. After a binding plateau was achieved it was incubated with PBST and the cuvette regenerated for the next experiment.

Immobilized SPA-- A 200-µl solution containing 20 µg of sGM-CSFRalpha -Fc in PBST was added to the cuvette and allowed to bind for 6 min (phase 1). PBST was added for 5 min (phase 2). GM-CSF at various concentrations in PBS was then added and allowed to bind for 5 min (association phase, phase 3) for 5 min. PBST was then added for an additional 5 min (dissociation phase, phase 4). No regeneration was performed between runs in these assays.

Data Analysis

Data were collected automatically and analyzed subsequently using the Microsoft Excel program (Microsoft, Redmond, WA). Plots were fitted using Cricket Graph (Cricket Software, Malvern, PA). The sGM-CSFRalpha -Fc surface prepared by capturing sGM-CSFRalpha -Fc on immobilized SPA was corrected for a downward drift as dissociation of sGM-CSFRalpha -Fc from the SPA occurred along with GM-CSF binding to sGM-CSFRalpha -Fc. Accordingly, control experiments were run in which sGM-CSFRalpha -Fc was allowed to bind to SPA for 6 min (phase 1), the receptor-associated surface was washed for 5 min (phase 2), and then phosphate-buffered saline was added for an additional 5 min (to simulate the association phase of GM-CSF, phase 3) followed by a final 5-min wash (simulating the dissociation phase of GM-CSF from sGM-CSFRalpha -Fc, phase 4). In these experiments, the negative slope of the dissociation of GM-CSFRalpha -Fc from SPA decreased slightly after each of the washes (in phases 2, 3, and 4). Therefore, correction for sGM-CSFRalpha -Fc dissociation from SPA was performed separately for each phase. The slope of dissociation of sGM-CSFRalpha -Fc from SPA in the final 200 s of phase 2 was calculated for each experiment and used to normalize this portion of the sensorgrams. Similarly, the slope of the final 200 s of phase 4 was calculated and used to correct the dissociation phase of GM-CSF from sGM-CSFRalpha -Fc. A weighted average of the slopes from phases 2 and 4 was used to predict a slope for phase 3, and this was used to correct the association phase of GM-CSF to the sGM-CSFRalpha -Fc. Evaluation of these corrections on the mock experiments gave reasonable agreement with the observed data (data not shown). This triphasic correction was used for each experiment with immobilized SPA.

Kinetic analysis was performed as described previously (23). In the case of a bimolecular interaction of two species A and B whose association and dissociation are regulated, respectively, by kon and koff for giving the final product AB,
<FR><NU>d[<UP>AB</UP>]</NU><DE>dt</DE></FR>=k<SUB><UP>on</UP></SUB>[<UP>A</UP>][<UP>B</UP>]−k<SUB><UP>off</UP></SUB>[<UP>AB</UP>]. (Eq. 1)
If B is immobilized on the cuvette, the response R is proportional to the amount of product AB, thus,
<FR><NU>dR</NU><DE>dt</DE></FR>=k<SUB><UP>on</UP></SUB>[<UP>A</UP>](R<SUB><UP>max</UP></SUB>−R)−k<SUB><UP>off</UP></SUB>R, (Eq. 2)
which rearranges to
<FR><NU>dR</NU><DE>dt</DE></FR>=k<SUB><UP>on</UP></SUB>[<UP>A</UP>]R<SUB><UP>max</UP></SUB>−(k<SUB><UP>on</UP></SUB>[<UP>A</UP>]+k<SUB><UP>off</UP></SUB>)R (Eq. 3)
where Rmax is the maximum response. Therefore, a plot of dR/dt versus R will have a slope of -(kon[A] + koff) = -ks, and a plot of ks versus [A] will have a slope of kon.

In the case of the dissociation phase, any free A from dissociation of the product is assumed to be removed by the buffer so that there is no reassociation ([A] = 0). Therefore,
<FR><NU>dR</NU><DE>dt</DE></FR>=<UP>−</UP>k<SUB><UP>off</UP></SUB>R, (Eq. 4)
hence
R=R<SUB>0</SUB>e<SUP><UP>−</UP>k<SUB><UP>off</UP></SUB>t</SUP>. (Eq. 5)
Therefore, the slope of a plot of ln(R0/R) versus time represents koff. In practice, reassociation may not be eliminated by the wash, and its effect may be detected in interactions characterized by fast-on fast-off kinetics such as this one. This can result in the ln(R0/R) versus time plots deviating from a straight line.

The equilibrium dissociation constant (KD) was calculated from the equation
K<SUB>D</SUB>=<FR><NU>k<SUB><UP>off</UP></SUB></NU><DE>k<SUB><UP>on</UP></SUB></DE></FR> (Eq. 6)
and from a plot of R/concentration versus R, analogous to a Scatchard plot of Bound/Free versus Bound, where the slope corresponds to -1/KD.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cloning of sGM-CSFRalpha -Fc

We had previously cloned a soluble form of the GM-CSFRalpha into a retroviral vector (12). We used this clone, which encodes for sGM-CSFRalpha , to prepare a fusion protein of the sGM-GM-CSFRalpha with the Fc portion of murine IgG. The sGM-CSFRalpha -Fc construct (shown in Fig. 1) was obtained as follows. sGM-CSFRalpha cDNA from clone 9 and the CH2-CH3 cDNA region of mouse anti-reovirus mAb 9BG5 (IgG2a) were PCR amplified using the primers described under "Materials and Methods." An analytical 2% agarose gel indicated a band of ~1,100 bp and a band of ~650 bp, respectively, for sGM-CSFRalpha and CH2-CH3. After purification on a 1% low melting temperature agarose gel the two products were fused in a PCR-SOEing step, obtaining a construct of ~1,700 bp. The construct was digested and ligated into the BamHI/SalI site of the pBabe-puro vector. After transformation of E. coli DH5alpha cells, a colony with the right sized insert was selected, grown, and DNA purified on a Qiagen column. The construct was verified by restriction enzyme mapping and sequencing as described under "Materials and Methods."


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Fig. 1.   sGM-CSFRalpha -Fc construct. The construct was cloned into the BamHI and SalI sites of pBabe-puro as reported under "Materials and Methods." SV40, simian virus 40; MuLV, murine leukemia virus; LTR, long terminal repeat.

Transfection and Expression of the Fusion Protein

sGM-CSFRalpha -Fc in the pBabe vector was transfected into a retroviral packaging cell line, and the virus-containing medium was used to transduce SP2/0 cells. These SP2/0 cells were selected and subcloned as described under "Materials and Methods." Clones were tested for the presence of the construct by reverse transcriptase PCR, with 9 of 96 clones expressing the sGM-CSFRalpha -Fc by this assay. Several subclones were isolated and grown to confluence; the selective media were removed and supernatants collected. These supernatants were screened for production of sGM-CSFRalpha -Fc by two different ELISAs as described under "Materials and Methods." An example of one ELISA is shown in Fig. 2, comparing the binding of clone supernatants with that of neutralizing anti-GM-CSF mAb 126.213 (24). In this example, the ELISA plate was coated with SPA to which the sGM-CSFRalpha -Fc was bound. Biotinylated GM-CSF was then added, and binding was detected by streptavidin-HRP conjugate. This assay shows significant binding of several clones to GM-CSF. Similar results were obtained in an assay in which the ELISA plate was coated with GM-CSF, then the sGM-CSFRalpha -Fc was bound and binding detected by anti-mouse Ig-HRP (data not shown). Clones that consistently displayed binding were chosen for further analysis.


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Fig. 2.   ELISA on clone supernatants. ELISA plates were coated with SPA followed by mAbs against GM-CSF or supernatants containing the sGM-CSFRalpha -Fc. Binding was detected by a biotinylated GM-CSF/avidin-HRP/TMB system. Panel A, a dilution curve of mAbs 126.213 (neutralizing anti-GM-CSF (24)) and 9BG5 (control anti-reovirus mAb (13)). Panel B, assay of supernatants from individual clones. The mean of duplicate wells is shown.

Purification and Characterization of sGM-CSFRalpha -Fc

sGM-CSFRalpha -Fc was purified from ~140 ml of clone supernatant on an SPA column, as described under "Materials and Methods." The partially purified sGM-CSFRalpha -Fc was analyzed in an 8% acrylamide gel (Fig. 3). In the gel run under oxidizing conditions there is a high molecular mass band of ~160-200 kDa, representing multimers of the fusion protein, whereas in the gel run under reducing conditions there are two main bands, at ~60 kDa and ~43 kDa (sGM-CSFRalpha -Fc monomer and uncharacterized contaminant, respectively). We scanned the gel using Image 1.41 (Wayne Rasband, NIMH, Bethesda, MD) and performed densitometry, allowing us to estimate that the sGM-CSFRalpha -Fc fusion protein represents ~50% of the total protein purified by the SPA column. Gel filtration chromatography (using a Sephacryl S-200HR column) was used to evaluate the molecular mass of the sGM-CSFRalpha -Fc multimer. The chromatogram indicated that the oxidized sGM-CSFRalpha -Fc runs at ~160 kDa (data not shown). When compared with the reducing SDS-polyacrylamide gel electrophoresis of the fusion protein, this suggests that the sGM-CSFRalpha -Fc forms trimers. We have seen the ~60-200-kDa form from several sGM-CSFRalpha -Fc preparations consistently and reduce to ~60 kDa upon reduction consistently.


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Fig. 3.   Size estimation of sGM-CSFRalpha -Fc using a gray scale reproduction of a Coomassie Blue-stained protein gel. 45 µg of protein purified from each of two clones was analyzed on an 8% acrylamide gel under oxidizing (lanes A and B) and reducing (lanes C and D) conditions. The corrected molecular masses of prestained molecular mass standards are also shown.

Western blot analysis with anti-human-GM-CSFR mAbs to detect the receptor portion of the fusion protein and anti-mouse-IgG to detect the Fc portion of the fusion protein indicated a high molecular mass band for the oxidized forms (sGM-CSFRalpha -Fc trimer) and a strong band at 60 kDa for the reduced form (sGM-CSFRalpha -Fc monomer) (Fig. 4).


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Fig. 4.   Western blot analysis of sGM-CSFRalpha -Fc. Western blots were performed using anti-GM-CSFRalpha mAbs or anti-mouse IgG as described under "Materials and Methods." In all membranes lane 1 is 75 µg of fusion protein, and lane 2 is a mixture of high molecular weight markers. Upper gels, detection by mAb anti-human-GM-CSFR (mouse IgM). Middle gels, detection by mAb anti-human-GM-CSFR (mouse IgG2a). Lower gels, detection by anti-mouse IgG. The secondary antibody alone (which is mouse Ig Fab-specific) showed no reactivity on similar blots (data not shown).

Binding Analysis of sGM-CSFRalpha -Fc

The SPA-purified sGM-CSFRalpha -Fc was evaluated next for binding to GM-CSF and for bioactivity. A competitive ELISA assay (Fig. 5) was carried out to confirm that the sGM-CSFRalpha -Fc bound to native GM-CSF. An ELISA plate was coated with SPA followed by the sGM-CSFRalpha -Fc. We then added unlabeled GM-CSF at various dilutions, followed by biotinylated GM-CSF, with binding detected by an avidin-HRP conjugate. We saw increasing binding with increasing amounts of biotinylated GM-CSF (compare the A450 nm without competitor for 7, 70, and 700 pM biotinylated GM-CSF), and this was inhibited competitively by increasing amounts of unlabeled GM-CSF. This indicates that unmodified GM-CSF binds to our fusion protein.


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Fig. 5.   Binding of unmodified GM-CSF to purified sGM-CSFRalpha -Fc fusion protein. SPA-coated ELISA wells were used to bind the sGM-CSFRalpha -Fc protein. After incubation with unmodified GM-CSF, biotinylated GM-CSF was added and the system developed with the avidin-HRP/TMB system as described under "Materials and Methods." The read-out is A450 nm, with the mean ± S.D. of triplicate wells shown.

A radiolabeled binding assay (Fig. 6) confirmed the binding of the sGM-CSFRalpha -Fc fusion protein to 125I-GM-CSF. In this experiment, increasing the amount of added fusion protein increased the binding 125I-GM-CSF, and this binding was blocked fully by excess cold GM-CSF. This confirms the binding of GM-CSF to the sGM-CSFRalpha -Fc fusion protein.


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Fig. 6.   Binding of radiolabeled GM-CSF to purified sGM-CSFRalpha -Fc fusion protein. On a radioimmunoassay plate coated with SPA, the fusion protein was added at different concentrations followed by 125I-GM-CSF (388 pM) with or without cold GM-CSF (100 nM). After 1 h at room temperature the plate was quenched on ice, washed, and counted. The mean ± S.D. counts/min (CPM) bound of triplicate values is shown for 125I-GM-CSF alone (Hot), 125I-GM-CSF plus cold GM-CSF (Hot+Cold), as well as the Delta CPM bound (hot - (hot + cold)).

Bioactivity of sGM-CSFRalpha -Fc

We next evaluated the ability of the sGM-CSFRalpha -Fc to inhibit the biological activity of GM-CSF using a GM-CSF-dependent cell line. MO7E cells are a myelomonocytic cell line and are dependent on GM-CSF for growth. We evaluated the growth of MO7E cells in the presence of GM-CSF with or without added sGM-CSFRalpha -Fc using the MTT assay (see "Materials and Methods"). The presence of increasing amounts of sGM-CSFRalpha -Fc resulted in increasing inhibition of proliferation of the MO7E cells (Fig. 7). We found that 250 µg/ml sGM-CSFRalpha -Fc produced 75% inhibition in cellular proliferation. In contrast, the sGM-CSFRalpha -Fc had no effect on the growth of the IL-2-dependent cell line CTLL (data not shown). This indicates that the sGM-CSFRalpha -Fc is a specific biological antagonist of GM-CSF.


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Fig. 7.   Bioactivity of sGM-CSFRalpha -Fc. The GM-CSF-dependent cell line MO7E was used to test the ability of sGM-CSFRalpha -Fc to block GM-CSF-dependent cell growth. 104 cells/well were incubated with various dilutions of sGM-CSFRalpha -Fc with or without 125 pM GM-CSF. After a 1-day incubation, proliferation was evaluated as noted under "Materials and Methods." The percent inhibition of proliferation ± the S.D. of triplicate wells is shown.

Biosensor Analysis

GM-CSF Binding to sGM-CSFRalpha -Fc-- The sGM-CSFRalpha -Fc was evaluated for the kinetics of binding using a biosensor. Initially, the binding of sGM-CSFRalpha -Fc to immobilized SPA was analyzed to assure that the Fc portion of our receptor was functional. A typical sensorgram obtained is shown in Fig. 8A. After the addition of sGM-CSFRalpha -Fc, a bulk phase effect is seen initially (increase in the response from bulk phase refractive index) followed by an association phase. With washing, an initial rapid response decrease (likely from bulk phase refractive index decrease) becomes relatively exponential. Fig. 8B shows a typical dR/dt plot for the association phase. The initial rapid response shift (likely bulk phase effect) is shown in open circles, followed by a relatively linear association shown in filled circles. The plot of dR/dt for the linear phase shows a ks of 2.51 × 10-3/s for this concentration of sGM-CSFRalpha -Fc.


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Fig. 8.   Kinetics of sGM-CSFRalpha -Fc binding to SPA. Panel A, typical sensorgram obtained for binding of sGM-CSFRalpha -Fc to immobilized SPA. The relative response (in relative units, RU) is shown versus time for 20 µg of sGM-CSFRalpha -Fc added at 85 s. The increase in relative units indicates binding of sGM-CSFRalpha -Fc to SPA. The sGM-CSFRalpha -Fc was washed at 445 s, beginning the dissociation phase of the experiment. Panel B, association rate constant calculation. A plot of dR/dt versus time is shown, as described under "Materials and Methods." An early rapid association phase (probably the result of mass effect) is shown by open circles, with a later slower association phase indicated by filled circles. A curve fitted to the second phase is shown, which has a slope (ks) = 2.51 × 10-3/s. Panel C, dissociation rate constant calculation. The dissociation phase is replotted as ln(R1/Rn) versus time. Data points are shown following a steep initial decline in relative units (see Fig. 9A), likely resulting from mass effect. The slope from the early data points (filled circles) was used to calculate a straight line with a slope corresponding to koff = 2.00 × 10-3/s.

Calculation of the off-rate (koff) is shown in Fig. 8C. Departure from linearity is seen, possibly caused by rebinding, which increases with time. The plot of ln(R1/Rn) versus time for the initial phase yields a koff of 2.00 × 10-3/s for this interaction. The relative linearity of the dissociation phase of sGM-CSFRalpha -Fc from SPA seen in Fig. 8A encouraged us to attempt to analyze a monovalent interaction of GM-CSF to the sGM-CSFRalpha -Fc bound to SPA. This was a result of our desire to analyze a monovalent interaction of GM-CSF with the binding sites on the sGM-CSFRalpha -Fc, as sGM-CSFRalpha is monovalent. We felt that the linearity of the dissociation of sGM-CSFRalpha -Fc from SPA would allow us to correct for this while analyzing the interaction of GM-CSF with the sGM-CSFRalpha -Fc.

For these experiments, a standardized protocol was developed. sGM-CSFRalpha -Fc was allowed to bind to SPA for 6 min (phase 1), followed by a 5-min wash (phase 2). GM-CSF was then added and allowed to bind (association phase, phase 3) for 5 min, followed by a final dissociation phase of 5 min (phase 4). Preliminary experiments performed with phosphate-buffered saline added in place of GM-CSF at the initiation of phase 3 revealed that the downward slopes of phases 2, 3, and 4 increased sequentially. We therefore corrected for the downward slopes of these three phases separately, as noted under "Materials and Methods." The data from the final 200 s of phases 2 and 4 were used to correct these phases. An intermediate slope was calculated for phase 3 and used to correct this phase.

A typical sensorgram, with the predicted "base lines," is shown in Fig. 9A. The values for the predicted base lines were subtracted from the data points in the sensorgram and yielded the corrected sensorgram shown in Fig. 9B. A relatively rapid initial association phase is apparent, which is supplanted by a more gradual association phase. dR/dt plots of these data were nonlinear (data not shown). Departure from the linearity expected for a single bimolecular interaction (Equation 3) could be caused by multiple modes of interaction with different affinities, cooperativity, or other complex models.


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Fig. 9.   Kinetics of GM-CSF binding to sGM-CSFRalpha -Fc bound to SPA. Panel A, typical sensorgram obtained for GM-CSF binding to sGM-CSFRalpha -Fc which was prebound to SPA, as in Fig. 8. 20 µg of sGM-CSFRalpha -Fc was bound to SPA for 6 min (as in Fig. 10A) at which point the sGM-CSFRalpha -Fc was washed out. GM-CSF binding was initiated 5 min after the initial wash-out of sGM-CSFRalpha -Fc. The 200 s before the addition of GM-CSF is shown. In this experiment, 800 nM GM-CSF was added. The association phase lasted 5 min, at which point the GM-CSF was washed out for an additional 5 min. Each circle indicates a data point. Two processes are evident: dissociation of sGM-CSFRalpha -Fc from SPA (the progressive down-slope), and the association/dissociation of GM-CSF with the sGM-CSFRalpha -Fc. The predicted base line (in fact, the continuous dissociation of sGM-CSFRalpha -Fc from SPA) was determined as noted under "Materials and Methods" and is shown for the three phases of the experiment (before GM-CSF was added, the association phase, and the dissociation phase). Panel B, the same sensorgram shown in Fig. 10A corrected for dissociation of sGM-CSFRalpha -Fc from SPA. The values for the predicted base line were subtracted from the data points shown in Fig. 8A to produce this corrected sensorgram. Panels C-F, dissociation rate constant calculations for 400 nM (C), 800 nM (D), 1,600 nM (E), and 3,200 nM GM-CSF (F). The dissociation phase of the corrected sensorgrams (as shown in panel B) were replotted as the ln(R1/Rn) versus time. Data points are shown after a steep initial decline in RU (see Fig. 10B), likely resulting from mass effect. A line fitted to the data was calculated, with a slope corresponding to the koff values, which were 2.53 × 10-2/s (C), 2.34 × 10-2/s (D), 2.32 × 10-2/s (E), and 2.54 × 10-2/s (F).

Analysis of the dissociation phase is shown in Fig. 9, C-F. The relative linearity of the plot of ln(R1/Rn) versus time was seen for several concentrations of GM-CSF, including 400, 800, 1,600, and 3,200 nM. This linear relationship was maintained for at least the first 100 s of the dissociation phase. The calculated koff was remarkably stable for these different experiments, ranging from 2.32 × 10-2 to 2.54 × 10-2/s (average 2.43 ± 0.12 × 10-2/s).

Immobilized GM-CSF-- The binding of various concentrations of the sGM-CSFRalpha -Fc trimer to immobilized GM-CSF was measured using the conditions described under "Materials and Methods." Fig. 10A shows an overlay of sensorgrams obtained with 75, 300, 600, and 900 nM sGM-CSFRalpha -Fc. After an association phase showing the binding of sGM-CSFRalpha -Fc to immobilized GM-CSF, a washing step with buffer alone was used to effect the dissociation phase.


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Fig. 10.   Panel A, sensorgrams obtained showing the binding of various concentrations of sGM-CSFRalpha -Fc to immobilized GM-CSF. The numbers indicate the concentrations of sGM-CSFRalpha -Fc added in each run. At 100 s the buffer used to equilibrate the cuvette is replaced by sGM-CSFRalpha -Fc solution. The increase in response indicates the binding of sGM-CSFRalpha -Fc to the immobilized GM-CSF (association phase). All association phases are run to the steady state to plot a Scatchard plot. After the steady state is reached, the ligand solution is replaced by the buffer (in this figure, to overlay the sensorgrams, we normalized that time point at 675 s). The decay of response represents the dissociation of bound sGM-CSFRalpha -Fc (dissociation phase). Panel B, association rate constant calculation. The association phases of sensorgrams were plotted as the slope of the initial region of the curve at a given time versus the relative response at that time. The slopes of these lines give values for ks at each concentration (see "Materials and Methods"). The data points used to calculate ks are filled; the other points are not. Panel C, dissociation rate constant determination. The dissociation phase of the sensorgram at highest sGM-CSFRalpha -Fc concentration in Fig. 8A was replotted as ln(R1/Rn) (natural log of the response at time zero of dissociation/response at time n) versus time (see "Materials and Methods"). The curve shows the analysis of the dissociation phase following the association of 900 nM sGM-CSFRalpha -Fc to the immobilized GM-CSF. The straight line shows the line fit to the first 100 s of the data (filled-in data points). The slope of this line gives a dissociation rate constant (koff) of 1.57 × 10-3/s.

The association phase of each sensorgram was analyzed by plotting the change in response over time (dR/dt) versus the response (R) according to Equation 3. The dR/dt plots, shown in Fig. 10B, were nonlinear, with at least two phases apparent. This departure from linearity was similar to that seen in experiments with sGM-CSFRalpha -Fc bound to immobilized SPA, suggesting a complex process irrespective of the assay orientation.

The dissociation phase was analyzed by assuming that it represents the irreversible release of sGM-CSFRalpha -Fc and so should follow an exponential decay. Data were plotted as the ln(response at time zero of dissociation/response at time n) versus time according to Equation 5 (Fig. 10C). Again, nonlinearity was observed, but the initial ~140 s were quite linear, so rates were calculated for these time points as has been reported previously (25). The dissociation rate constant (koff) was calculated as the slope of these plots. For this calculation, the highest concentration of sGM-CSFRalpha -Fc was used because this concentration gave the most accurate dissociation curve. The value of koff was calculated as 1.57 × 10-3/s-1. This indicates a slower off-rate for this experimental configuration than that shown in Fig. 9, C-F (see above), probably because of the multivalent nature of sGM-CSFRalpha -Fc.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this paper we report the preparation and characterization of a fusion protein obtained by expression of a construct made from sGM-CSFRalpha and the Fc portion of a mouse antibody. The fusion protein is produced easily by transduced cells, recovered from their medium, and purified on an SPA column via the Fc portion. This protein should provide a useful means for the study of the interaction of GM-CSF or GM-CSF mimics with the GM-CSFRalpha .

After amplification and SOEing of the sGM-CSFRalpha cDNA and the CH2-CH3 region of mouse anti-reovirus mAb 9BG5 (IgG2a), a construct of the expected size was obtained and ligated into pBabe-puro vector (Fig. 1). This construct was used to produce recombinant retrovirus, which was used to transduce SP2/0 cells. After subcloning the sGM-CSFRalpha -Fc-transduced SP2/0 cells, supernatants were tested for their binding to biotinylated GM-CSF. For all supernatants we obtained low values in the ELISAs (Fig. 2 and data not shown), probably due in part to the low concentration of fusion protein in the supernatants and in part to the rapid off-rate of GM-CSF (see below). However, several positive clones were obtained which allowed more extensive analysis of purified sGM-CSFRalpha -Fc.

Denaturing polyacrylamide gel analysis of SPA-purified sGM-CSFRalpha -Fc under reducing and nonreducing conditions indicated a similar pattern of molecular masses for sGM-CSFRalpha -Fc purified from two different clones (Fig. 3). Moreover, both polyacrylamide gel electrophoresis under nonreducing conditions and gel filtration chromatography of the purified oxidized protein indicated that the fusion protein was present as a multimer, probably a trimer of ~160-200 kDa. SDS-polyacrylamide gel electrophoresis performed under reducing conditions indicated a ~60-kDa protein (Fig. 3), which represented ~50% of the total protein in the preparation. Also seen was a ~45 kDa band (which was absent in other preparations) and a smaller band of ~25 kDa which is seen at the dye front of the gel in Fig. 3. A gel run in the same conditions alongside fetal calf serum as a control shows bands of ~60 and ~25 kDa in the fetal calf serum (data not shown). Thus, it is likely that there is antibody contamination from the fetal calf serum used to grow our sGM-CSFRalpha -Fc-transduced SP2/0 cells. Bovine IgG under oxidizing conditions likely co-migrates with the multimeric sGM-CSFRalpha -Fc, with the bovine IgG heavy chain also co-migrating with the sGM-CSFRalpha -Fc under reducing conditions. Alternatively, the other bands seen on the reducing gel could represent proteins that covalently attach to the sGM-CSFRalpha -Fc. We think this is unlikely as the quantity of contaminating proteins varies in our different preparations. In addition, a two-dimensional gel run under reducing conditions showed a single band with an estimated pI of 6.7 (data not shown).

The nature of the ~60 kDa band was investigated by Western blot analysis, using antibodies against the GM-CSFRalpha and murine IgG. The experiment indicated that the ~60 kDa band is detected by two different anti-human GM-CSFR monoclonal antibodies, recognizing the receptor portion of the fusion protein, and by anti-mouse IgG, recognizing the Fc portion of the sGM-CSFRalpha -Fc fusion protein (Fig. 4). The other contaminating bands were not recognized by either reagent, making it unlikely that these represent breakdown products.

This biochemical and immunochemical analysis strongly suggests that the sGM-CSFRalpha -Fc exists as a trimer in the oxidized state. The reason for this is unclear. The fusion protein has a total of 16 cysteines: 12 from the sGM-CSFRalpha and 4 from the IgG2a Fc sequence. These are thought to participate in intramolecular disulfide bridges in native GM-CSFRalpha and murine IgG, respectively. However, some of these cysteines appear to form intermolecular disulfide bridges in the sGM-CSFRalpha -Fc. We are uncertain of the disulfide bonding pattern of the sGM-CSFRalpha -Fc which results in trimer formation. It is interesting to speculate that such trimers of the GM-CSFRalpha may exist naturally, possibly through noncovalent interactions. However, no data to support or refute this possibility are currently available. The trimers seen here show that trimer formation can occur without abrogating binding of the sGM-CSFRalpha -Fc to GM-CSF. We are uncertain if this is an artifact of the sGM-CSFRalpha -Fc construct or a reflection of a physiologic capability for receptor aggregation to occur.

The binding capacity of the fusion protein for GM-CSF was tested in several experiments. Competition of "cold" and biotinylated GM-CSF on an ELISA plate coated with sGM-CSFRalpha -Fc indicated that the fusion protein specifically binds to GM-CSF (Fig. 5). This binding was also confirmed by a radioligand binding assay, using 125I-GM-CSF (Fig. 6), which again showed specific binding that was abrogated by cold GM-CSF. This indicates that the fusion protein possesses active binding site(s) for GM-CSF and that this binding is specific as it is inhibited competitively by unlabeled GM-CSF. It was also of interest to determine whether the sGM-CSFRalpha -Fc was active in a bioassay. Because the sGM-CSFRalpha -Fc binds GM-CSF, it would be expected to block binding of GM-CSF to cell surface receptors. This indeed appears to be the case, as the sGM-CSFRalpha -Fc also prevented proliferation of the GM-CSF-dependent cell line MO7E when present in the medium together with the growth factor (Fig. 7). Thus, the sGM-CSFRalpha -Fc is similar to sGM-CSFRalpha , as we described previously (12), in possessing biological antagonist activity.

The parameters regulating the association and dissociation between GM-CSF and sGM-CSFRalpha -Fc were evaluated in preliminary studies of the binding of GM-CSF to sGM-CSFRalpha -Fc in turn bound to immobilized SPA using an IAsys optical biosensor (Figs. 8 and 9). This was only possible because of the relatively linear dissociation of sGM-CSFRalpha -Fc from SPA, as shown in Fig. 8. The advantage of this experimental design is that GM-CSF exists as a monomer in solution, and a 1:1 interaction with a single receptor site is likely. This eliminates the problem posed by the trimeric nature of the sGM-CSFRalpha -Fc, which may contribute an avidity component to binding. The disadvantage is that a complicated correction for the dissociation of sGM-CSFRalpha -Fc from SPA was needed. In these experiments, association phase analysis revealed nonlinear plots of dR/dt versus R (data not shown). Although the reason for this departure from linearity is obscure, given this experimental configuration, it is unlikely to be the result of the trimer formation by sGM-CSFRalpha -Fc. This prevented formal kon calculations. We were able to obtain reproducible values for koff which averaged 2.43 × 10-2/s (Fig. 9, C-F). When used to solve for t1/2 in Equation 4 for the case of r = 1/2 R0, the estimated half-time for receptor dissociation ranges from 27 to 30 s.

Similar analyses were carried out for immobilized GM-CSF with the sGM-CSFRalpha -Fc in solution. The association phase failed to follow the predicted theoretical model (23) as demonstrated by the plots of both dR/dt versus R and ks versus concentration. In the dR/dt versus R plot (Fig. 10B) we represented the first linear portion of the curves for 75, 300, and 600 nM concentrations, but we omitted from the plot the 900 nM concentration. This high sGM-CSFRalpha -Fc concentration resulted in very rapid binding in the initial association phase (Fig. 10A). It was therefore impossible to obtain enough data points to draw a reliable dR/dt versus R plot for this concentration. Moreover, with such a rapid association rate, mass transfer becomes the rate-limiting factor in the initial stage of the binding. We therefore declined to calculate a formal kon from these data.

For analysis of the dissociation phase, we chose to analyze only the plot for the highest concentration of sGM-CSFRalpha -Fc, 900 nM, because this had the highest response/background ratio. This analysis did not fit the expected exponential decays shown by the curvature of the plot in Fig. 10C. Nonlinearity in dissociation phase analysis has also been seen in the interaction of insulin-like growth factor and insulin-like growth factor-binding protein (26). In this case the effect was attributed to either long term maturation of the analyte-ligand complex or functional heterogeneity of the immobilized ligand. Other potential explanations include multiple sites with different affinities, cooperativity in binding, rebinding, and mass effects. Curve-fitting the data in Fig. 10C showed a fit to the sum of exponentials, and this is perhaps the result of differential binding characteristics of sites within the sGM-CSFRalpha -Fc trimer, depending on the number of active receptor sites already involved in binding to immobilized GM-CSF. In the absence of a definite physical explanation the data were analyzed by taking the slope of the curve in the first 120 s. In support of this method of analysis are its simplicity and the consistency of the calculated koff with the average value from experiments with different sGM-CSFRalpha -Fc concentrations. The koff value determined by this method is 1.57 × 10-3/s. This is ~1 order of magnitude slower than for the reverse experimental configuration (see Fig. 9, C-F). This indicates that the trimer formation of sGM-CSFRalpha -Fc slows the off-rate, likely because of an avidity effect.

Thus, our data indicate that the sGM-CSFRalpha -Fc interaction with GM-CSF is characterized by a rapid dissociation phase, implying that the major energy of binding is contributed by a fast on-rate. These kinetics have relevance to the biological activity of the sGM-CSFRalpha . The ability of the sGM-CSFRalpha to function as a biological antagonist is likely caused by competition for free GM-CSF with the transmembrane form of the receptor (tmGM-CSFRalpha ) present on cells. Given the relatively fast off-rate of the sGM-CSFRalpha -Fc, the antagonist activity of sGM-CSFRalpha is then dependent on its continued presence, because once the sGM-CSFRalpha diffuses away, the GM-CSF would dissociate and then be available for binding to the tmGM-CSFRalpha . As noted above, the t1/2 for dissociation is on the order of 27-30 s. Thus, for sGM-CSFRalpha to sequester GM-CSF and remove it from the local environment of a responsive cell, diffusion away from the cell would have to be more rapid than this dissociation rate. Slower rates of diffusion would imply that the sGM-CSFRalpha needs to be present in the vicinity of the cell to act as a competitive inhibitor.

In terms of understanding the high affinity sites formed by tmGM-CSFRalpha and beta c, these data would suggest that the contribution of beta c is mostly to slow the off-rate, as the on-rate is already relatively rapid. This is supported by experiments reported by Gearing et al. (4), where binding of 125I-GM-CSF to low affinity sites on HL-60 cells was lost after a short (10-min) incubation in the absence of 125I-GM-CSF, but the high affinity sites remained. We would postulate, based on our data and theirs, that the primary role of the GM-CSFRalpha in the complex is to capture GM-CSF with a rapid association phase and that the beta c functions primarily to slow the dissociation phase. It is interesting to speculate on the role of the beta -subunit (beta c) in slowing the dissociation of GM-CSF from the heterodimeric receptor. Studies of GM-CSF mutants support a key role for Glu-21 in binding to the high affinity but not the low affinity receptor (8, 27-31). These authors would argue for direct binding of the GM-CSF A-helix, centered on residue Glu-21, to the beta c. Such direct binding would account for the slower off-rate seen with the heterodimeric receptor. However, direct evidence is lacking for such an interaction, and conformational changes or aggregation of the GM-CSFRalpha imparted by beta c could also account for the slower off-rate seen. Clarification of this matter awaits direct binding data with GM-CSFRalpha , beta c, and GM-CSF.

When compared with data from other cytokine receptor interactions, the kinetics of binding for GM-CSF to the sGM-CSFRalpha -Fc are similar to those reported for other cytokines, such as IL-5 (25). The characteristics of rapid on-rates and somewhat slower, but still rapid, off-rates may be a general characteristic of cytokine-receptor interactions for four-helix bundle cytokines.

    ACKNOWLEDGEMENTS

We thank Joann Kwah, Christine Myers, and Joan VonFeldt for technical and intellectual assistance and J. C. for inspiration.

    FOOTNOTES

* This study was supported by grants from the American Cancer Society and the Arthritis Foundation (to W. V. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Grants R01 AI/GM 36502 and AI/GM 40462.

§ To whom correspondence should be addressed: 913 Stellar-Chance Laboratories, University of Pennsylvania School of Medicine, 422 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-662-2789; Fax: 215-349-5572; E-mail: rheum{at}sunspark.med.upenn.edu.

1 The abbreviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; GM-CSFR, GM-CSF receptor; sGM-CSF, soluble GM-CSF; tmGM-CSF, transmembrane GM-CSF; IL, interleukin; PCR, polymerase chain reaction; mAb, monoclonal antibody; SOE, splicing by overlap extension; bp, base pairs; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; SPA, staphylococcal protein A; HRP, horseradish peroxidase; TMB, 3,3',5',5'-tetramethylbenzidine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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