A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor alpha chains

Abstract

Two series of peptides that specifically bind to the extracellular domain of the alpha chain of the human interleukin-5 receptor (IL-5Ralpha), but share no primary sequence homology to IL-5, were identified from libraries of random recombinant peptides. Affinity maturation procedures generated a 19-aa peptide that binds to the IL-5 receptor alpha/beta heterodimer complex with an affinity equal to that of IL-5 and is a potent and specific antagonist of IL-5 activity in a human eosinophil adhesion assay. The active form of the peptide is a disulfide-crosslinked dimer that forms spontaneously in solution. Gel filtration analysis, receptor-binding studies, and analytical ultracentrifugation reveal that the dimeric peptide binds simultaneously to two receptor alpha chains in solution. Furthermore, the dimer peptide, but not IL-5, can activate a chimeric receptor consisting of the IL-5Ralpha extracellular domain fused to the intracellular domain of the epidermal growth factor receptor, thus demonstrating that the peptide also promotes receptor dimerization in a cellular context. The functional antagonism produced by the bivalent interaction of the dimeric peptide with two IL-5R alpha chains represents a distinctive mechanism for the antagonism of cytokines that use heteromeric receptors.

Figure 1

Peptide sequences and structures. Amino acid residues are indicated with single letter code. Sequences of clones 1 and 2 determined by DNA sequencing. AF-numbered compounds were prepared synthetically and their structures were confirmed by MS. In the AF17121 sequence, (Ac) and (NH₂) indicate acetylation and amidation, respectively. Boldface and underlined letters C in the sequences of clone 1 and clone 2 indicate the invariant cysteine residues specified in the library design. Letter S in AF11875, AF17121, and AF18748 structures indicates cysteine sulfur involved in the disulfide bonds shown. Letters SH in AF17362 structure indicate free cysteine sulfhydryls.

Figure 2

Structure determination of dimeric AF17362 by tryptic digestion and MS. Aliquots from a tryptic digest of the dimer form of AF17362 were removed every hour and analyzed by MS. Shown are the molecular masses (in daltons) of all of the species observed (not all were observed in a single time point). The structures shown, along with their calculated molecular masses, are the only ones consistent with the observed molecular masses, the sequence of AF17362, and the cleavage specificity of trypsin.

Figure 3

Pharmacological characterization of antagonist peptides. (A) Competition-binding assay using TF-1 cells. The binding of ¹²⁵I-labeled human IL-5 to TF-1 erythroleukemia cells was reduced by competition with various concentrations of human IL-5 (○), AF17121 (□), or AF18748 (●). Results are expressed as percentage of maximum specific binding (% B/B₀)_. Mean total binding was 1,074 ± 188 cpm and nonspecific binding was 115 ± 20 cpm. Data are mean ± SEM for three separate experiments for IL-5 and AF18748, and mean of two separate experiments for AF17121. (B) IL-5-induced eosinophil activation assay. The adherence of purified human eosinophils to IgG-coated plates in response to stimulation with 20 pM IL-5 was assessed in the presence of various concentrations of AF17121 (▴) or AF18748 (●). Data are mean ± SEM for four separate experiments for AF17121 and for six separate experiments for AF18748. The average OD₄₉₀ reading for IL-5-stimulated cells was 0.90 ± 0.09 compared to unstimulated levels of 0.17 ± 0.03. (C) AF18748 specificity assay. Purified eosinophils were assayed for their ability to adhere to IgG-coated plates after stimulation with 6 pM human GM-CSF, 180 pM human IL-3, 1 nM human tumor necrosis factor-α , or 100 nM fMet-Leu-Phe in the presence (closed bars) or absence (open bars) of 1 μM AF18748. The concentration of each cytokine used is one that elicits 80% of the maximal response achievable with that cytokine. Data are mean ± SEM for three separate experiments.

Figure 4

Biochemical evidence of AF18748-induced IL-5Rα dimerization. (A) Microvolume fluorimetry. Fluorescently labeled IL-5Rα ECD was incubated with IL-5Rα ECD-coated polystyrene beads and serial dilutions of AF18748 (●), AF17121 (○), or human IL-5 (□). Binding reactions were incubated in microtiter plates overnight at room temperature, and the bead-associated fluorescence was determined by using an FMAT instrument (Perkin–Elmer). Data points represent averages of triplicate determinations from a single representative experiment. (B) Gel filtration chromatography. IL-5Rα ECD was loaded onto a Precision Superdex 200 3.2/30 column alone (dotted line) or in the presence of equimolar amounts of AF17121 (light line) or AF18748 (dark line). Peak I corresponds to free AF17121 (2.5 kDa), peak II to free AF18748 (3.9 kDa), peak III is where monomeric IL-5R ECD elutes (89.5 kDa), and peak IV corresponds to a molecular mass of 209 kDa. Results shown are from a single experiment, representative of three separate runs for each condition. (C) Analytical ultracentrifugation. IL-5Rα ECD was examined alone (Upper) or in the presence of equimolar amounts of AF18748 in both reference and sample compartments (Lower). Data were analyzed by using both ultrascan and dcdt. Data are shown as the distribution of species observed, overlaid with curves fitted to a Gaussian distribution (Upper) or two overlapping Gaussian distributions (Lower). Data shown are from a single experiment representative of three.

Figure 5

Chimeric IL-5Rα/EGFR-reporter cell assay. (A) AF18748 induced activation of the IL-5Rα/EGFR chimeric receptor. Ba/F3 cells expressing a chimeric IL-5Rα/EGFR and containing a luciferase reporter gene were treated with serial dilutions of AF18748 (●), AF17121 (○), or IL-5 (□). Data points represent mean ± SEM for three separate experiments performed in triplicate. The data has been normalized to the stimulation induced by 1 μM AF18748 in each experiment. (B) Inhibition of AF18748-induced activation of the IL-5Rα/EGFR chimeric receptor by IL-5. Cells were treated with 200 pM AF18748 in the presence of increasing concentrations of IL-5. Data points represent the average of triplicate determinations from a single representative experiment.