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International Immunology, Vol. 11, No. 12, 1935-1944, December 1999
© 1999 Japanese Society for Immunology

Mapping and characterization of the epitope(s) of Sch 55700, a humanized mAb, that inhibits human IL-5

Ji Zhang, Reshma Kuvelkar, Nicholas J. Murgolo, S. Shane Taremi, Chuan-Chu Chou, Peng Wang, Motasim M. Billah and Robert W. Egan

Schering-Plough Research Institute, K-15C113/1600, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA

Correspondence to: J. Zhang


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
mAb against human IL-5 inhibit pulmonary eosinophilia, tissue damage and airway hyper-reactivity in allergic animal models. Sch 55700 is a humanized, neutralizing anti-IL-5 antibody. To better understand the molecular mechanism by which Sch 55700 blocks IL-5 bioactivity, we have mapped its epitope by scanning IL-5 with synthetic peptides. Those peptides containing a region, ERRRV, corresponding to amino acids 89–93 of IL-5 specifically interact with both Sch 55700 and its parental rat IgG, 39D10. Among the five residues of this region, all three arginine residues were particularly critical for interaction of these peptides with Sch 55700. We further characterized this region by alanine scanning using site-directed mutagenesis. Examination of COS-expressed IL-5 mutants by Western blot showed that single mutations of E89, R90, R91 or R92 to alanine caused a loss of IL-5 binding to both Sch 55700 and 39D10. We further demonstrated in surface plasmon resonance studies using a BIAcore biosenosor that E89, R90 or R91 are involved in the interaction between IL-5 and its receptor {alpha} subunit. Based upon the findings here and previously reported structures of the IL-5 and 39D10 variable region, we propose a model suggesting that the molecular interactions between the IL-5 and Sch 55700 mainly involve several ion pair interactions. We conclude that Sch 55700 occupies a region, ERRR, on IL-5 that is essential for its interaction with the receptor and thereby blocks IL-5 bioactivity.

Keywords: asthma, cytokine, eosinophil, molecular modeling, peptide scan


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Human IL-5, a disulfide-linked homodimeric glycoprotein, is a T cell-derived cytokine (1–3). The IL-5 crystal structure elucidated a novel two-domain structure in which each domain adopts a four {alpha} helix bundle structure (A–D) (4–5). This IL-5 fold is similar to many other cytokines including IL-2 (6), IL-4 (7), granulocyte-macrophage colony-stimulating factor (GM-CSF) (8) and macrophage colony-stimulating factor (9). While all the other cytokines are monomeric, the dimeric topology of IL-5 is unique in that each bundle is composed of three helices from one monomer and a fourth helix contributed by the second monomer (4).

The biological effects of IL-5 are mediated through its binding to a heterodimeric receptor complex composed of {alpha} and ß subunits. While the {alpha} subunit is responsible for IL-5 binding specificity, the ß subunit is shared by IL-3 and GM-CSF, and is essential for both the formation of the high-affinity receptor complex and the signal transduction (10). The pathophysiologic role of IL-5 is based upon our understanding that this cytokine selectively enhances eosinophil production and release from bone marrow, chemotaxis, activation and survival (11–12). The eosinophilia is closely associated with chronic inflammatory conditions such as asthma, rhinitis and atopic dermatitis (3,11–13). Thus, blocking the action of IL-5 may provide therapeutic benefit in these allergic disorders. Indeed, administration of neutralizing anti-IL-5 mAb to mouse, guinea pig or primate models of allergic asthma inhibits the development of airway eosinophilia and bronchial hyper-reactivity (14–17). This and many other studies have confirmed that the eosinophil, and more specifically IL-5, is an attractive target for therapeutic intervention in allergic diseases such as asthma. Therefore, it would seem possible to treat humans with anti-IL-5 neutralizing antibodies to attenuate the lung eosinophilia and the decrease in lung function that occur during asthma.

39D10, a rat mAb with a Kd of 53 pM against human IL-5, is a neutralizing antibody that blocks the IL-5-dependent proliferation of the human erythroleukemic cell line, TF-1 (18). To develop it into a therapeutic antibody, 39D10 was humanized using complementarity determining region grafting technology into a human framework with a {kappa} light chain and a {gamma}4 constant region (19,20). The humanized antibody, designated Sch 55700, was shown to retain the potency of the parent antibody by blocking IL-5 receptor (IL-5R) binding and by inhibiting IL-5-induced cell proliferation (20,21), suggesting that the conformation of antigen recognition in 39D10 was properly restored in Sch 55700. Furthermore, Sch 55700 was found to effectively inhibit eosinophilic inflammation and airway hyper-responsiveness in mice, guinea pigs, rabbits and monkeys (21,22).

To understand how antibodies such as 39D10 and Sch 55700 block the interactions between IL-5 and IL-5R, it is essential to delineate the epitope(s) of the antibodies. In the studies described in this report, we found a small region, ERRR, corresponding to amino acids 89–92 of IL-5, that was critical for both Sch 55700 and 39D10 to specifically interact with IL-5. We further show in surface plasmon resonance (SPR) studies using a BIAcore biosenosor that E89, R90 or R91 are involved in the interaction between IL-5 and its receptor {alpha} subunit. Based upon these findings and the known crystal structures of both IL-5 and the 39 D10 variable region, we propose a topological model of the molecular interactions between the IL-5 and Sch 55700. We conclude that Sch 55700 occupies a region, ERRR, on IL-5 that is essential for its interaction with the receptor and thereby blocks IL-5 bioactivity.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents
Human IL-5 and Sch 55700 (recombinant humanized 39D10 antibody) batch no. 5-aIL5-07, were provided by Schering-Plough (Union, NJ). Rat anti-hIL-5 mAb, 39D10 and rat anti-mIL-5 mAb TRFK-5 were supplied by John Abrams at DNAX Research Institute of Molecular and Cellular Biology (Palo Alto, CA) and Michael Minnicozzi at Schering-Plough Research Institute respectively. Both anti-hIL-5 neutralizing mAb (mouse) and polyclonal antibody (goat) were purchased from R & D Systems (Minneapolis, MN). Mouse anti-hIL-5Rß subunit mAb was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Baculovirus-expressed soluble human IL-5R{alpha} and rat IgG2a were provided by Chuan-Chu Chou (Schering-Plough Research Institute). Human IgG4 from myeloma plasma was purchased from Athens Research and Technology (Athens, GA). Human IL-5 cDNA expression vector (pcDSR{alpha} HuIL5) was provided by Kim Stranick (Schering-Plough Research Institute).

Peptide scan analysis
The cellulose-bound peptides arrayed as sets of peptide scan membranes were obtained from Jerini Bio Tools (Berlin, Germany). Different peptides were synthesized on single cellulose membrane supports according to spot synthesis technology developed by Kramer et al. (23). For peptide scanning of the IL-5 molecule, 51 different 13mer peptides with 11 residues overlapping and covering the whole amino acid sequence of IL-5 (24) were synthesized and immobilized on 8x1.5 cm cellulose paper. Each spot represented one of 51 peptides and the spots were arranged in a 3x20 format to have 20 columns and three rows. For mutational analysis, 260 13mer peptides were synthesized and immobilized on a 8x5.5 cm cellulose paper. Each spot represented one of 260 peptides and spots were arranged in 20 columns and 13 rows. All spots in the first column were wild-type peptides with an amino acid sequence of KSGEERRRVNQFL. From columns 2 to 20, the spots contained the mutant peptides in which each residue of KSGEERRRVNQFL was replaced by one of 19 amino acids (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y) respectively. Visualization of antibody binding to peptide(s) on cellulose paper was performed by the following procedures. The peptide spot paper was first incubated in methanol for 10 min at room temperature, then washed 3 times with TBS buffer (0.05 M Tris–HCl, pH 8.0 and 0.14 M NaCl). The peptide spot paper was further incubated for 1 h in blocking buffer (5% non-fat dry milk; 0.05 g/ml sucrose and 0.05% Tween/TBS). The tested antibody was diluted with blocking buffer to a final concentration of 5 µg/ml, then incubated with the peptide paper for 3 h at room temperature with gentle shaking. After removing the solution containing the test antibody, horseradish peroxidase-labeled anti-human, anti-rat or anti-mouse IgG (Boehringer Mannheim, Indianapolis, IN) was added at 1:4000 dilution with blocking buffer. Following a 1 h incubation at room temperature, the antibody solution was removed and the peptide spot paper was washed 3 times with TBS buffer. The detection was then performed using the BM Chemiluminescence Blotting Substrate (POD) detection kit (Boehringer Mannheim). The films were developed and evaluated by attributing the spots to their corresponding peptides in the sequence list, which is supplied with the peptide spot paper.

Site-directed mutagenesis and mutant IL-5 expression.
Eight site-directed mutants of IL-5 cDNA were generated from a human IL-5 cDNA expression vector, pcDSR{alpha} HuIL5, using various mutant oligonucleotides (see Table 1Go) and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Specific substitutions of 1 or 2 bp at different sites resulted in a change of a codon into one for A (alanine) (see Fig. 4Go). All mutants were confirmed by double-strand DNA sequencing. For expression of mutants, COS-7 cells were seeded at 1x105/well (a diameter of 3.5 cm) and transfected with 1 µg plasmid DNA using LipofectAMINE Plus Reagent (Gibco/BRL, Gaithersburg, MD).


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  		Table 1. Oligonucleotide primers used in site-directed mutagenesisa
 


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  		Fig. 4. Western blot analysis of COS-expressed IL-5 mutants. The IL-5 mutants G87(M1), E88(M2), E89(M3), R90(M4), R91(M5), R92(M6), V93(M7) and N94(M8), and the wild-type protein (Wt) were harvested from the supernatants of transfected COS cells that transiently expressed these proteins. After precipitation by 5% TCA, the samples were analyzed by 12% SDS–PAGE and electroblotting onto nitrocellulose membranes. Both the wild-type and the mutants were visualized by different anti-hIL-5 antibodies including polyclonal IgG (anti-hIL-5), Sch 55700, 39D10, TRFK-5 and R 7D anti-hIL-5 mAb, followed by chemiluminescence. The detailed experimental procedures are described in Methods.

 
Western blot analysis
The supernatants were collected 3 days after transfections and precipitated by adding 5% TCA. After incubation on ice for 15 min, the samples were centrifuged at 16,000 g for 15 min. The pellets were washed with cold acetone, then neutralized by 5 µl of 1 M Tris–HCl buffer (pH 8.0). The pellets were further dissolved in a 100 µl final volume by adding a non-reduced 3xSDS–PAGE sample loading buffer (0.125 M Tris–HCl, pH 6.8, 20% glycerol, 4% SDS and 0.005% bromophenol blue) with a brief sonication. Then 5 µl of each precipitated COS-expressed protein was resolved by electrophoresis on 12% PAGE–SDS minigel (Novex, San Diego, CA), then electroblotted to nitrocellulose membranes using the Novex system and a blotting buffer containing 20% methanol. Membranes were blocked by overnight incubation at 4°C in PBS/5% dry milk/0.1% Tween 20. Membranes were then incubated for 1 h at room temperature in 10 ml PBS buffer containing 0.1% Tween 20, 1% fetal bovine serum and one of several anti-hIL-5 antibodies: Sch 55700 (260 µg), 39D10 (16 µg), TRFK-5 (129.2 µg), R & D mouse anti-hIL-5 monoclonal (5 µg) and R & D goat anti-hIL-5 polyclonal (20 µg). After washing with PBS/0.1% Tween 20 4 times, membranes were incubated with one of the following horseradish peroxidase-labeled anti-IgG antibodies including anti-human, anti-rat and anti-mouse IgG polyclonal F(ab') fragments (Boehringer Mannheim) for 1 h. For detection of goat anti-hIL-5 polyclonal antibody, membranes were first incubated with a biotin-labeled anti-goat IgG antibody (Amersham, Arlington Heights, IL) for 1 h, then an additional 1 h with horseradish peroxidase-labeled avidin (Amersham). The COS-expressed IL-5 mutants were detected using the ECL Western blotting detection kit (Amersham). A pre-stained protein standard, SeeBlue Pre-Stained Standard, was purchased from Novex (cat. no. LC5625) and used as a protein size marker.

SPR measurements
The molecular interaction between IL-5 and baculovirus-expressed soluble human IL-5R{alpha} chain was determined by SPR measurements using the BIAcore instrument (Pharmacia Biosensor, Piscataway, NJ) described in details elsewhere (25,26). Both IL-5R{alpha} and IL-5 were immobilized on a sensor chip surface using amine coupling reagents kit provided by the manufacturer, with slight modifications of the available procedure. The eluent buffer was 50 mM HEPES, pH 7.4, 0.15 M NaCl, 3.4 mM EDTA and 0.005% P20. All experiments were carried out at 25°C and at a flow rate of 5 µl/min or as indicated otherwise.

To monitor the interactions of IL-5 mutants with the soluble IL-5R{alpha}, the carboxymethylated dextran surface of a sensor chip was activated with a 25 µl injection of NHS (N-hydroxysuccinimide)/EDC [N-ethyl-N'-(dimethylaminopropyl)carbodiimide] followed by injection of 20 µl of Sf9-derived soluble IL-5R{alpha} at 20 µg/ml in 10 mM sodium acetate, pH 4.0. The remaining activated esters were blocked by injection of 30 µl of 1 M ethanolamine. These conditions resulted in 3080 response units, corresponding to 1.5 mM of soluble IL-5R{alpha}, immobilized on the chip. Then 40 µl of each COS-expressed IL-5 wild-type, mutant and media alone were injected onto the immobilized IL-5R chip at a flow rate of 5 µl/min. The sensor chip was regenerated successfully between each run with a 1 min pulse of 500 mM sodium carbonate, pH 10.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Mapping of Sch 55700 epitope
To map the region(s) of IL-5 that are recognized by Sch 55700 and other antibodies, a number of 13mer peptides spanning the whole IL-5 sequence and overlapping each other by 11 amino acids were synthesized on a cellulose paper support (24). This peptide spot paper was probed by the antibodies to test their binding activities and detected by chemiluminescence. Peptides 40, 41, 42, 43 and 44 interacted with both Sch 55700 and 39D10 but not with the other antibodies, suggesting that these peptides contained a binding site specific to both Sch 55700 and 39D10 (Fig. 1AGo). A consensus sequence, ERRRV (Fig. 1BGo), was identified among these peptides corresponding to amino acids 89–93 of human IL-5.




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  		Fig. 1. Mapping of Sch 55700 binding epitope on IL-5. (A) Peptide scan assay. Fifty-one 13mer peptides with 11 overlapping residues scanning the whole IL-5 sequence (24) were synthesized and immobilized on cellulose paper. The spots were arranged in a 3x20 format. The cellulose papers were incubated with 39D10, Sch 55700, rat IgG2a, human IgG4 and mouse mAb (anti-hIL-5Rß) respectively. The peptides that bind these antibodies were visualized by incubation with corresponding horseradish peroxidase-labeled secondary antibodies (anti-IgG antibody) and a chemiluminescence detection method. The spots indicated by 40, 41, 42, 43 and 44 were bound to both Sch 55700 and 39D10. The detailed experimental procedures are described in Methods. (B) A consensus sequence of Sch 55700 binding peptides 40, 41, 42, 43 and 44.

 
To further characterize the essential nature of this region for Sch 55700 binding, mutational analysis was performed with a 13mer peptide spanning amino acids 85–97 of human IL-5. Residues were individually replaced by one of 19 different amino acids, resulting in 260 peptides on the cellulose paper shown in Fig. 2Go. All three arginines (R) were found to be essential for Sch 55700 binding, because any substitution abolishes its interaction with this peptide (Fig. 2Go). Glutamic acid (E) at position 89 was also important, because substitutions except for phenylalanine (F), isoleucine (I), leucine (L) and tryptophan (W) either eliminated or decreased Sch 55700 binding (Fig. 2Go). This result suggests that the ERRR motif appears to be a dominant contact region to interact with Sch 55700. However, the replacement of some amino acids at the periphery of the ERRR motif also had a significant impact on Sch 55700 binding. For example, replacing K85{uparrow} or G87{uparrow} to more hydrophobic amino acids (K85 replaced by F, I, L, S, W or Y; G87 replaced by F, I, L, P or W) enhanced Sch 55700 binding (Fig. 2Go). A change of V94{uparrow} to an acidic residue, D or E, led to an even more dramatic increase in Sch 55700 binding (Fig. 2Go). Thus, these data suggest that other amino acids on this 13mer peptide may also contribute to overall Sch 55700 binding. The replacement of R90, R91 or R92 by K that has a very similar ionic charge to R failed to retain Sch 55700 binding (Fig. 2Go), suggesting that the structure specificity of R is also critical for Sch 55700 to interact with the ERRR motif.



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  		Fig. 2. Mutational analysis of the Sch 55700 binding peptide KSGEERRRVNQFL. Each residue of the peptide was substituted by all 19 L-amino acids (rows) and analyzed for Sch 55700 binding. Each spot except the wild-type spots (control) in the first column represents a single substitution analogue. Sch 55700 binding was detected using procedures described in Fig. 1Go. The detailed experimental procedures are described in Methods.

 
89ERRR92 is an antigenic epitope for Sch 55700 binding to IL-5
To determine whether the region 89ERRR92 is essential for Sch 55700 binding to IL-5, eight site-directed mutants of IL-5 were constructed in which each residue from 87 to 94 of IL-5 was changed to A (alanine) (Fig. 3Go). Following transient expression in COS cells, these IL-5 mutants were isolated from the medium of cell culture and analyzed by Western blot analysis using several anti-hIL-5 antibodies. An anti-hIL-5 polyclonal IgG could detect all IL-5 mutants with similar intensities, suggesting that the expression levels of these mutants were comparable (Fig. 4Go). In contrast, neither Sch 55700 nor 39D10 could detect IL-5 mutants altered at E89, R90, R91 or R92 sites (Fig. 4Go), confirming that the linear region, 89ERRR92, on IL-5 is indeed an antigenic epitope for these antibodies. Interestingly, mutations at several sites near or within the 89ERRR92 region also had effects on two other antibodies that neutralize hIL-5, TRFK-5 and R & D mouse monoclonal IgG. For example, a change at E89, R91, R92 or N94 abolished the binding of TRFK-5 to IL-5 (Fig. 4Go) and a mutation at R91, R92 or V93 decreased the binding of the R & D antibody to IL-5 (Fig. 4Go). The fact that residues R91 and R92 are shared by the epitopes of independent IL-5-neutralizing antibodies strongly suggest a critical role of this region for the biological activity of IL-5.



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  		Fig. 3. Schematic illustration of site-directed IL-5 mutants altered at the 87GEERRRVN94 of IL-5. Each residue of 87GEERRRVN94 was replaced with A (alanine), generating eight single-site-directed IL-5 mutants named M1(G), M2(E), M3(E), M4(R), M5(R), M6(R), M7(V) and M8(N).

 
89ERRR92 is critical for IL-5 to bind IL-5R{alpha}
The biological responses to IL-5 are mediated by its cell surface receptor (10). Because the R91 and R92 residues are either essential or important for IL-5 to be recognized by four neutralizing mAb reported in Fig. 4Go, the 89ERRR92 region may be a binding site to the IL-5 receptor. To test this hypothesis, COS-expressed IL-5 mutants were evaluated for their ability to bind IL-5R{alpha}, the IL-5 specific subunit (10), using the BIAcore system. Purified soluble IL-5R was immobilized on the surface of a CM chip using standard amine coupling and IL-5 binding was standardized by injecting a known amount of standard purified human IL-5 onto the surface. Next, equal amounts of each COS-expressed IL-5 wild-type, mutant or media alone were injected onto the immobilized IL-5R chip. The total amounts of bound IL-5 for each run was determined by measuring the total number of response units 10 s after the injection (Fig. 5Go). The total amount of bound IL-5 mutants altered at G87 (M1), E88 (M2), V93 (M7) or N94 (M8) were not affected as compared to the wild-type protein (Fig. 5Go). In contrast, the binding to IL-5R of IL-5 mutants altered at E89 (M3), R90 (M4), R91 (M5) and R92 (M6) was reduced. Least amount of binding was observed for the IL-5 mutant, R91 (M5). This result suggests that the region 89ERRR92 of IL-5 is also an important IL-5R{alpha} binding epitope and plays a critical role in the formation of the IL-5–IL-5R{alpha} complex.



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  		Fig. 5. Binding of COS-expressed IL-5 mutants to immobilized IL-5R{alpha} on BIAcore chips. The COS-expressed IL-5 mutants were analyzed by a polyclonal anti-hIL-5 antibody (Fig. 4Go), quantified and normalized using an Agfa Studiostar scanner. Equal amounts of each COS-expressed IL-5 wild-type, mutant or media alone were injected onto the chip containing immobilized soluble IL-5R{alpha}. The total amounts of bound IL-5 for each run were determined by measuring the total number of response units 10 s after the injection. The mutants M1, M2, M3, M4, M5, M6, M7 and M8 represent G87, E88, E89, R90, R91, R92, V93 and N94 sites altered by A (alanine) (see Fig. 3Go) respectively. The detailed experimental procedures are described in Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The purpose of the studies reported here was to map the region of IL-5 which binds Sch 55700 and to better understand the mechanism by which Sch 55700 neutralizes IL-5 bioactivity. We have shown that a small region on IL-5 is critical for Sch 55700 binding. This region contains five amino acids, ERRRV, corresponding to amino acids 89–93 of IL-5. It was initially identified from a number of overlapping peptides that bound specifically to both Sch 55700 and its parental rat IgG, 39D10. The critical residues in this region were further identified by mutational analysis of a peptide in which each residue was separately replaced by one of 19 different amino acids and by site-directed mutagenesis of IL-5 cDNA.

When these IL-5 mutants were tested for their ability to bind IL-5R{alpha}, any single mutation within the 89ERRR92 region either abolished or decreased the interaction between IL-5 and its receptor. Therefore, it appears that Sch 55700 recognize a small region in IL-5 that is directly involved in its receptor binding. These results corroborate previous mutagenesis studies that showed a domain of the IL-5 dimer containing E89, R90 and R91 is critical for IL-5 binding to its receptor {alpha} (27–29). Therefore, Sch 55700 appears to neutralize IL-5 activity by directly occupying its receptor binding site.

The crystal structure of human IL-5 has a dimeric core of two four-helix bundles formed by two identical polypeptide chains joined covalently by disulfide bonds (Fig. 6Go) (4–5). By helix swapping, in which helix D from one chain combines with helices A, B, and C from the second, the IL-5 dimer is a duplex of four-helix bundle structures (30). In this IL-5 tertiary structure, 89ERRRV93, is localized in a loop region that links helices C and D (Fig. 6Go). Among the amino acids 89–94 of IL-5, all the residues are exposed to solvent with the exception of V93 (4), and can readily serve as binding epitopes for both Sch 55700 and IL-5R{alpha} subunit.



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  		Fig. 6. Structure of hIL-5 as determined by X-ray crystallography (4), showing the location of 89ERRR92. The IL-5 is an interdigitating homodimer, with four helices labeled A–D. One monomer is shown in purple and the other monomer is shown in green. Within the purple monomer, the residues G87, E88, E89, R90, R91, R92, V93 and N94 are illustrated in a small red region.

 
The Fab fragment of 39D10, a rat antibody from which Sch 55700 was constructed, has been crystallized (31). However, attempts to produce suitable crystals of the 39D10 or Sch 55700 Fab–IL-5 complex have, to date, been unsuccessful. We, therefore, attempted to dock the available antibody and antigen structures based on the epitope mapping data (Fig. 7AGo). The structures were docked manually by monitoring molecular contacts through use of the InsightII 97.0 program (MSI, San Diego, CA) on a Silicon Graphics O2 Workstation. Based upon the molecular modeling, key ion pair and hydrogen bond interactions have been identified and are summarized in Table 2Go. A resulting model of the complex suggested that the majority of the antibody antigen interactions were ion pairs (Fig. 7BGo). These ion pairs are E89(IL-5)–K93(39D10 light chain), R90(IL-5)–D58(39D10 heavy chain), R91(IL-5)–D56(39D10 heavy chain), R92(IL-5)–D98(39D10 heavy chain). While other residues may also be involved peripherally in high-affinity binding, the four residues, 89ERRR92, on IL-5 have been implicated as essential for Sch 55700 to bind and block IL-5 bioactivity.




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  		Fig. 7. Molecular modeling diagram illustrating the molecular detail of the 39D19 and IL-5 interactions. (A) The IL-5 and 39D10 interaction. The top purple/blue dimer is IL-5. The bottom is Fab 39D10 in which the heavy chain is shown in brown and the light chain is shown in blue. (B) The structural diagram showing the interaction regions and residues between IL-5 and Fab 39D10. The top part is the interaction region of IL-5 and the bottom is the interaction region of Fab 39D10. The several putative pair interactions between IL-5 residues and Fab 39D10 residues are indicated by double headed arrows.

 

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  		Table 2. Summary of key ion pair and hydrogen bond interactions
 
In comparison to Sch 55700, two other IL-5-neutralizing mAb, TRFK-5 and R & D antibody, were also tested for their abilities to bind IL-5 mutant proteins. From these studies it is clear that several residues both within and near the 89ERRR92 region on IL-5 are also involved in the epitopes of the other neutralizing antibodies. For instance, IL-5 mutants altered at E89, R91, R92 or N94 lost binding to TRFK-5. In contrast, the mutations at R90 or V93 have no effect on the binding of IL-5 to this antibody (Fig. 4Go). Changes at R91, R92 or V93 also decreased the binding of the R & D antibody to IL-5. These results suggest that the epitopes of three IL-5 neutralizing antibodies, Sch 55700, TRFK-5 and the R & D antibody, are distinct but overlapping. Because R91 and R92 are critical for IL-5 to bind Sch 55700, TRFK-5 and the R & D antibody as well as to its receptor {alpha}, directly occupying the receptor binding site appears to be a common mechanism by which three neutralizing antibodies block the biological activity of IL-5.

In summary, we have defined a small region, 89ERRR92, on IL-5 that is critical for IL-5's interaction with Sch 55700 and its receptor {alpha} subunit. Molecular modeling using the published structure data for both 39D10, a rat version of Sch 55700, and IL-5 suggested that four ion pairs form a central point of Sch 55700-IL-5 interaction. We conclude that Sch 55700 occupies a region, ERRR, on IL-5 that is essential for its interaction with the receptor and, thereby, blocks IL-5 bioactivity. This molecular detail of the Sch 55700 and IL-5 interactions will also be valuable to design small molecules that may have therapeutic effects in diseases that involve IL-5.


   Acknowledgments
 
We thank Dr Lothar Germeroth, Jerini Bio Tools GmbH in Berlin of German, for providing the IL-5 peptide spot papers and technical support.


   Abbreviations
 
GM-CSF granulocyte macrophage colony stimulating factor
IL-5R IL-5 receptor
SPR surface plasmon resonance

   Notes
 
Transmitting editor: M. Feldmann

Received 17 June 1999, accepted 19 August 1999.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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