Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.

We have synthesized a chemically defined, dimeric form of an erythropoietin mimetic peptide (EMP) that displays 100-fold increased affinity for the erythropoietin receptor (EPOR) and correspondingly elevated potency in cell-based assays and in mice. The dimeric EMP1 was synthesized using a C-terminal lysine residue as a branch point. A beta-alanine residue was coupled to the main-chain (alpha) amino group of the lysine residue in order to provide a pseudosymmetrical scaffold where both the side-chain and main-chain were of approximately equal length. Using an orthogonal protection system, independently disulphide-cylized EMP1 moieties were synthesized upon this scaffold. The proposed mechanism of increased potency of the dimer over the parental compound EMP1 is consistent with the structure of a cocrystal of EMP1 and the extracellular domain of the EPOR in which a noncovalent peptide dimer is seen spanning the cleft between two molecules of the EPOR extracellular domain.

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin 1 (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-la to the type I IL-1 receptor with IC5o values of 45-140 ,uM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.

Two series of peptides that specifically bind to the extracellular domain of the ␣ chain of the human interleukin-5 receptor (IL-5R␣), 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 ␣͞␤ 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 ␣ chains in solution. Furthermore, the dimer peptide, but not IL-5, can activate a chimeric receptor consisting of the IL-5R␣ 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 ␣ chains represents a distinctive mechanism for the antagonism of cytokines that use heteromeric receptors.I nterleukin-5 (IL-5) is a T cell-derived hematopoietic cytokine that acts exclusively on cells of the eosinophil and basophil lineage (1, 2). Although IL-5 can regulate many of the functions of mature, terminally differentiated eosinophils such as cell survival (3), adhesion (4), and activation (5), its primary function is to promote the differentiation and expansion of eosinophil precursors in the bone marrow (6).Transgenic mice that constitutively overexpress IL-5 in their lungs exhibit systemic and airway eosinophilia, bronchial hyperreactivity, and histopathological features characteristic of human asthma (7). Administration of recombinant IL-5 into the airways of either guinea pigs (8) or mildly asthmatic individuals (9) promotes a lung eosinophilia and bronchial hyperreactivity. Moreover, blocking the activity of IL-5 through administration of neutralizing anti-IL-5 antibodies (10-12), or through IL-5 gene deletion (13), prevents allergen-induced airway eosinophilia and bronchial hyperreactivity. Together, these data demonstrate that IL-5 plays a central role in the development of allergen-induced eosinophilia in animals and suggest that an anti-IL-5 therapeutic could provide an alternative approach to the treatment of human asthma perhaps more specific than current anti-inf lammatory therapies such as inhaled corticosteroids.The IL-5 receptor is a heterodimer consisting of an ␣ chain that specifically binds IL-5 and a signal-transducing ␤ c chain shared with the receptors for two structurally related cytokines: granulocyte-macrophage colony-stimulating factor (GM-CSF) and . A number of protein-based IL-5 antagonists have been reported, including soluble IL-5 receptor ␣ chains (IL5R␣s) that sequester the ligand in solution (15), single point mutants of IL...

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