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WO1999047151A1 - Ligands peptidiques pour recepteur d'erythropoietine - Google Patents

Ligands peptidiques pour recepteur d'erythropoietine Download PDF

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Publication number
WO1999047151A1
WO1999047151A1 PCT/US1999/005842 US9905842W WO9947151A1 WO 1999047151 A1 WO1999047151 A1 WO 1999047151A1 US 9905842 W US9905842 W US 9905842W WO 9947151 A1 WO9947151 A1 WO 9947151A1
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Prior art keywords
seq
isolated polypeptide
amino acid
neutral
peptide
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Stephen J. Mcconnell
Dominic G. Spinella
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Chugai Pharmaceutical Co Ltd
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Chugai Pharmaceutical Co Ltd
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Priority to AU31892/99A priority Critical patent/AU3189299A/en
Priority to JP2000536391A priority patent/JP4361684B2/ja
Priority to US09/646,691 priority patent/US6642353B1/en
Publication of WO1999047151A1 publication Critical patent/WO1999047151A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the fields of pha ⁇ nacology and drug discovery. More particularly, the invention relates to novel peptide compositions that can bind and activate the human erythropoietin receptor, and to methods of making small molecule agonists of the erythropoietin receptor using such peptide compositions as design templates.
  • Erythropoietin is a cytokine that stimulates the formation of red blood cells by inducing the growth and differentiation of progenitor cells.
  • the recombinant version of human EPO is a valuable therapeutic agent useful for treating anemia that is associated with several pathological conditions, including chronic renal failure, malignancy or the effects of chemotherapy, HIV and rheumatoid arthritis.
  • EPO When used therapeutically, EPO must be administered either by intravenous or subcutaneous injection. The fact that EPO is a relatively large glycoprotein adversely impacts the cost of manufacture, the pharmacological properties of molecule, and the mode of delivery of this therapeutic agent.
  • the erythropoietin receptor belongs to tfte cyto-ane receptor superfamily whose members share common structural features including an extracellular ligand binding domain, a single transmembrane-spanning region, and an intracellular cytoplasmic domain.
  • the extracellular domain (ECD) is sufficient to mediate receptor-ligand binding. It is therefore possible through recombinant DNA techniques to synthesize DNA encoding the ECD as a fusion with secreted proteins to produce reagents useful for identifying receptor binding molecules, for example by screening a phage display library.
  • Phage display libraries expressing fusions of random or semi-random peptides and bacteriophage coat proteins represent convenient versions of combinatorial libraries that can be screened to identify receptor ligands.
  • the random polypeptides Upon infection and assembly of phage particles, the random polypeptides are outwardly disposed for interaction with antibodies or other receptor probes. Since the phage particles contain the nucleic acid that encodes the fusion protein, the genetic information which identifies the sequence of the fusion protein is physically linked to the fusion protein. Construction and screening of such phage expression libraries are well known and have been described previously, such as, for example, in Sawyer et ⁇ , Protein Engineering 4:947-953 (1991); Akamatsu et ⁇ l., J. Immunol.
  • a soluble form of the EPOR was used to probe a phage display library to identify candidate peptides having EPO-like properties.
  • Wrighton et al. in Science 273:458 (1996) described the use of a fusion protein comprising the EPOR extracellular domain and human placental alkaline phosphatase in a library screening protocol.
  • the library used for this pu ⁇ ose displayed cyclic 8-residue peptides as fusions with the pNi ⁇ coat protein of a filamentous phage.
  • Peptides having higher affinity for the EPOR were subsequently isolated from mutagenesis libraries that displayed pHI protein fusions. This approach led to the identification of several peptides that stimulated erythropoiesis in mice. These agonists were disulfide-bonded cyclic peptides having the minimum consensus sequence
  • an isolated polypeptide capable of binding to a human erythropoietin receptor having the formula:
  • X Marinacea-C-X r X 2 -G-W-N-G-X 3 -C-X 4 -X 5 -W-X c wherein X tract is an amino-teimin ⁇ peptide of from 2 to 4 natural ⁇ -amino acids in length; X c is a carboxy-terminal dipeptide; and X,, X 2 , X 3 , X 4 and X 5 are independently selected from the group consisting of natural ⁇ -amino acids.
  • the amino-terminal X n is X ⁇ -X- 2 -X. X. ⁇ , wherein X abuse, is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural ⁇ -amino acids, or optionally X nl is absent; X transit 2 is selected from the group consisting of neutral and polar, neutral and hydrophobic, and basic natural ⁇ -amino acids, or optionally X n2 is absent if X nl is absent; X n3 is selected from the group consisting of natural ⁇ -amino acids; and X n4 is selected from the group consisting of neutral and polar, neutral and hydrophobic, and acidic natural ⁇ -amino acids.
  • X nl is E, G, ⁇ , S, D, Q, L, Y or A;
  • X n2 is F, N, A, K, R, G, S, I or L;
  • X n3 is H, Q, E, G, D, A, or N;
  • X n4 is E, G, N, A, P, D, T or M.
  • X nl is absent;
  • X pharmaceutical 2 is F, N, A, K, R, G, S, I or L;
  • X shelter 3 is H,
  • X nl is an acidic amino acid.
  • X n2 is a branched-chain amino acid or K.
  • X n3 is an acidic amino acid or N.
  • X n4 is N or G.
  • X is a neutral and hydrophobic, neutral and polar, or basic amino acid; more preferably X, is R, I, G, Q, L, T or S; and most preferably, is R or I.
  • X 2 is a neutral and hydrophobic, neutral and polar, or basic amino acid; more preferably, X 2 is R, P, W, G, L or ⁇ ; and most preferably is R.
  • X 3 is a neutral and polar, or basic amino acid; and more preferably X 3 is H, Q or ⁇ .
  • X 4 is a neutral and polar, basic, or acidic amino acid; more preferably X 4 is Q, ⁇ , S, K or E; and most preferably is K or ⁇ .
  • X 5 is a neutral and polar, neutral and hydrophobic, or acidic amino acid; more preferably is N, A, Y, D or E; and most preferably is D or E.
  • the carboxy-terminal X c is X cl - X,. 2 and
  • X cl is a neutral and polar, or neutral and hydrophobic amino acid
  • X c2 is a neutral and polar, neutral and hydrophobic, or basic amino acid.
  • X cl is L, I, P, F, M, Q or G; and more preferably is L or Q.
  • X c2 is M, W, T, S, G, N or R; and, more preferably, X c2 is G or R.
  • Other aspects of the invention are isolated polypeptides having the amino acid sequences of SEQ ED NO:81 and SEQ ID NO:82.
  • a method of activating a human erythropoietin receptor comprising the steps of contacting a cell having an erythropoietin receptor on it surface with a peptide mimetic of erythropoietin, wherein the peptide mimetic is a compound having the general formula
  • X n is an amino-terminal peptide of from 2 to 4 natural ⁇ -amino acids in length
  • X c is a carboxy-terminal dipeptide
  • X 1? X 2 , X 3 , X 4 and X 5 are independently selected from the group consisting of natural ⁇ -amino acids; and allowing the peptide mimetic to bind to the erythropoietin receptor, thereby initiating activation of the erythropoietin receptor.
  • Another aspect of the invention that is disclosed is a method of inhibiting binding of erythropoietin to an erythropoietin receptor, comprising the steps of providing a peptide mimetic having the general formula X n -C-X 1 -X 2 -G-W-N-G-X 3 - C-X 4 -X 5 -W-X c wherein X n is an amino-terminal peptide of from 2 to 4 natural ⁇ -amino acids in length; X c is a carboxy-terminal dipeptide; and X,, X 2 , X 3 , X 4 and X 5 are independently selected from the group consisting of natural ⁇ -amino acids, in sufficient quantity to compete with e.rythropoietin for binding to an erythropoietin receptor; and allowing the peptide mimetic to interact with the erythropoietin receptor, thereby inhibiting binding of erythrop
  • Another aspect of the invention is a method of discovering drugs that mimic erytliropoietin, comprising the steps of constructing a phage display library in which a fusion protein comprising a peptide consisting of a random sequence of amino acids and a phage protein; screening the phage display library for at least one clone that binds to a human erythropoietin receptor probe; isolating the clone that bind to a human erythropoietin receptor probe; determining a nucleic acid sequence from the clone that
  • the synthesizing step includes synthesizing a compound that is an organic compound, preferably a peptide. More preferably, the synthesized peptide is a cyclic peptide.
  • FIG. 1 is a line graph showing results from an EPOR-specific phage ELISA which confirmed that the ERB 1 phage clone specifically binds the chimeric Ig-EPOR probe.
  • the graph shows binding to Ig-EPOR as measured by an ELISA (O.D.) at different phage dilutions for a phage clone (ERB 1, *) isolated using the Ig-EPOR probe; for a phage clone (HPKF, ) that binds specifically to an anti-IL-8 antibody, and for a phage clone (IL6-B26, ⁇ ) that binds specifically to the human IL-6 receptor.
  • FIG. 2 is a line graph showing the results of an ELISA (O.D.) in which recombinant EPO at different concentrations (Units/ml) competed with the ERB 1 phage
  • FIGS. 3A and 3B show the approach that was used to create an ERB 1 evolved library.
  • FIGS. 3A and 3B show the approach that was used to create an ERB 1 evolved library.
  • FIGS. 3A and 3B show the approach that was used to create an ERB 1 evolved library.
  • FIGG. 3 A shows the amino acid sequence of the peptide that was fused to the p ⁇ l protein in the ERB 1 clone on the first line and the sequences of a collection of redundant olignucleotides that were used to create the evolved library of the ERB 1 clone on the second and third lines.
  • the oligonucleotides have been aligned to show the codons corresponding to the amino acid in the ERB 1 peptide and to show annealing of the oligonucleotides using 9 complementary bases at the 3' ends of the sequences.
  • 3B shows the nucleotide compositions that were used to synthesize the redundant oligonucleotides, for each of the bases represented as "g", "a”, “t”,”c”and “s” in the redundant oligonucleotides. Depicted are the amino acid sequence of the peptide that was fiised to the pin protein in the ERB1 clone and the scheme that was used to create a collection of oligonucleotides that encoded variants of the ERB 1 peptide sequence. .
  • FIG. 4 shows the amino acid sequences of phage clones derived from the ERB 1 evolved library and relative affinities of the clones for Ig-EPOR.
  • Clones Bl to B-21 were isolated by binding to Ig-EPOR, and phage clones Nl to N13 were derived from the evolved library but did not bind to Ig-EPOR during the selection process.
  • the relative affinities are numerical summaries of the data presented in FIGS. 5A to 5D.
  • FIGS. 5 A to 5D are a series of line graphs showing the affinities of several independent phage clones for the Ig-EPOR, relative to the original ERB 1 isolate.
  • FIG. 5 A shows results for clones B 1 to B5 and ERB 1 ;
  • FIG. 5B shows results for clones B6 to BIO and ERB1;
  • FIG. 5C shows results for clones Bl 1 to B15 and ERB1; and
  • 5D shows results for clones B16 to B21 and ERB1.
  • FIG. 6 is a line graph showing the results from a peptide inhibition ELISA in which binding of the chimeric Ig-EPOR probe to immobilized EPO was inhibited by competitor peptides (peptide ERB 1 -7, ⁇ ; peptide ERB 1 -8, •) compared to no peptide added ( ⁇ ) or addition of an irrelevant peptide that binds to IL-6 receptor (x).
  • competitor peptides peptide ERB 1 -7, ⁇ ; peptide ERB 1 -8, •
  • FIG. 7 is a line graph showing binding of the ERB 1-7 18-mer peptide ( ⁇ ) to Ig- EPOR and compared to binding to Ig-EPOR of N-terminal truncated ERB 1-7 peptides (17-mer, ⁇ ; 16-mer, *; 15-mer, x; 14-mer, *), and compared to addition of an irrelevant peptide that binds to IL-6 receptor (•) or no inhibitor added ( +).
  • FIGS. 8 A and 8B are line graphs showing the results from a TF-1 cell proliferation assays.
  • FIG. 8A shows a dose response curve for proliferation of TF-1 cells (O.D. at 450 nm) at different concentrations (Units) of recombinant EPO; and
  • FIG. 8B shows a dose response curve for proliferation of TF-1 cells (O.D. at 450 nm) at different concentrations ( ⁇ M) of the synthetic peptides ERB 1-7 ( ⁇ ) and ERB 1-8 (*), compared to addition of an irrelevant peptide that binds to IL-6 receptor (x).
  • EPOR and methods of making small molecule analogs of EPO using peptide ligands as design templates are unrelated to the primary amino acid sequence of the authentic EPO protein.
  • the peptides disclosed herein also are unrelated to previously isolated EPOR binding peptides from phage display libraries.
  • novel peptides identified herein are unrelated to previously isolated EPOR binding peptides from phage display libraries.
  • -6- serve not only as candidate therapeutic agents, but also as models for designing small molecules having EPO agonist or antagonist activities.
  • peptides or other small molecules that mimic the biological properties of EPO would represent valuable additions to the pharmacopeia.
  • a collection of cyclic peptides that bind the EPOR and mimic the pharmacological activity of erythropoietin were identified.
  • One EPOR-binding clone was initially isolated from an M13 phage library displaying 38 random amino acids fused to the N-terminus of the pin capsid protein.
  • Two exemplary synthetic peptides having amino acid sequences based on the consensus 12-mer sequence, inhibited binding of authentic EPO to a chimeric Ig-EPOR in an in vitro assay. Moreover, these derivative peptides also stimulated proliferation of an EPO-responsive cell line in culture. Truncating one of the 18-mer synthetic peptides by one or more N-terminal residues decreased the peptide activity in receptor- binding and biological assays.
  • EPOR ligands disclosed herein were identified by a multi-step procedure that involved: (1) creating a chimeric receptor probe that binds EPO; (2) creating a phage display library expressing a large number of target random peptides; (3) screening the library with the chimeric receptor probe to identify one or more lead
  • the use of a probe that included the ligand binding domain of the native receptor allowed binding of structures as large as the native ligand or as small as the minimal critical structure needed for receptor binding.
  • phage display libraries offer a rich source or moiecuiar uiversny wnicn can be rapidly screened using high throughput screening .assays.
  • the libraries employed in the screening procedures disclosed below displayed fusions with the pin minor coat protein of phage M13. While the N-terminal domain of the pm molecule binds the bacterial F pilus and is required for infection, the C- te ⁇ ninal domain anchors the protein in the phage capsid.
  • fusions of a large range of sizes can be tolerated in the constructions that involve the pin protein, thereby enhancing the molecular diversity of the library.
  • Relatively long random amino acid sequences e.g., about 30 to about 50 residues
  • Libraries of long random peptides offer a "sliding window" of adjacent amino acid residues, and also have the potential of providing tertiary structures that represent discontinuous epitopes in complex proteins. This added level of complexity may be important when screening for molecules capable of interacting with complex targets such as cell surface receptors. Moreover, libraries of long random peptides make possible the formation of multiple contact sites that are capable of binding complex targets. Production and Testing of Derivatives of Lead Peptides
  • this process of creating libraries of variant peptides based on the nucleic acid sequence coding for a lead peptide is referred to as "molecular evolution.”
  • molecular evolution this process of creating mutant clones having increased ability to interact with a receptor of interest (i.e., clones with increased binding affinity for the EPOR relative to that of the lead clone).
  • chimeric protein is meant a non-naturally occurring protein or polypeptide comprising some or all of the amino acid sequences from at least two different proteins or polypeptides, or of one protein or polypeptide and a non-naturally occurring polypeptide chain.
  • a chimeric protein is designed, constructed by genetic engineering, synthesized, or otherwise selected intentionally, and contains at least two domains (i.e., at least a first domain and a second domain, each having some structural and/or functional characteristic that is not present in the other domain).
  • a molecule may contain a label moiety which emits a signal which is capable of being detected directly (e.g., radioisotope, dye, or fluorescent or chemiluminescent moiety), or may contain a moiety which, through some additional reaction (i.e., indirectly), is capable of being detected (e.g., an attached enzyme, ligand such as biotin, enzyme substrate, epitope, or nucleotide sequence).
  • second molecule is meant a molecule which is able to bind to at least a portion of the second domain of a chimeric protein, thereby allowing detection or purification of the chimeric protein.
  • hinge region or “immunoglobulin heavy chain hinge region” is meant one of a family of proline-containing and cysteine-containing amino acid sequence regions which occur between the C H2 and C H1 regions of many mammalian immunoglobulin heavy chains, or analogs of these amino acid sequences based thereon, in which the regions to the amino- and carboxyl-terminal sides of the hinge are spatially separated by a turn or kink in the polypeptide chain, to facilitate separate and simultaneous specific binding to other molecules.
  • ligand is meant a molecule or a multimeric molecular complex which can specifically bind to another given molecule or molecular complex (i.e., its target).
  • a ligand is soluble while its target is immobilized, such as by an anchor domain imbedded into a cell membrane.
  • receptor is meant at least a portion of a molecule or a multimeric molecule complex wliich, in its native environment, has an anchor domain embedded into a cell membrane and is able to bind a given molecule or molecular complex. Often a receptor is capable of transducing an intracellular signal in response to ligand binding. Many receptors have particularly high affinity for a ligand when either or both the receptor or ligand are in a homomultimeric or heteromultimeric form (e.g., a dimer).
  • solid support an insoluble matrix either biological in nature, such as, without limitation, a cell or bacteriophage particle, or synthetic, such as, without limitation, an acrylamide derivative, cellulose, nylon, silica, and magnetic particles, to which soluble molecules may be linked or joined.
  • modified is meant non-naturally occurring or altered in a way that deviates from naturally-occurring compounds.
  • molecular evolution is meant a process of creating a library of variant peptides by randomization, at a controlled rate, of a nucleic acid sequence coding for a lead peptide having desired functional characteristics.
  • molecule is meant a molecular-sized inorganic or organic compound, such as, for example, a peptide, protein, nucleic acid, fat or fatty acid, which may be naturally ocurring or synthetically produced.
  • multimeric molecular complex is meant a complex comprising at least two molecular components, where the individual components may be, for example, a peptide, protein, nucleic acid, fat or fatty acid, where the complex is held together by covalent bonds, non-covalent bonds or other known chemical interactions.
  • Amino acids described by either their three letter (or one letter) abbreviations, are classified according to the nature of their side-groups (as described in Genes V, B.
  • neutral and hydrophobic which includes Ala (A), Nal (N), Leu (L), lie (I), Pro (P),
  • Branched-chain amino acids refers to I, L and V.
  • random peptides are displayed on the surface of bacteriophage M13 as N-terminal fusions expressed on major (e.g., pNIU) or minor (e.g., pill) coat proteins.
  • major e.g., pNIU
  • minor e.g., pill
  • Individual bacteriophage particles displaying sequences having desirable binding characteristics can be affinity purified and cloned using standard laboratory techniques.
  • Most phage libraries are constructed to display random sequences of only 6 to 8 amino acids in length as pDI or pNDI fusion proteins.
  • the first residue was an acidic, or neutral and polar, or neutral and hydrophobic amino acid, preferably D, E, G, N, S, Q, L, Y or A, and more preferably D.
  • the second residue was a basic, or neutral and polar, or neutral and hydrophobic amino acid, preferably L, V, I, K, A, F, R ,G or S, and more preferably a branched-chain amino acid, most preferably L.
  • the third residue was any of the four general types of amino acids characterized by the nature of their side chains, and preferably was E, D, Q, G, H, V or A, more preferably E or Q.
  • the fourth residue was an acidic, or neutral and polar, or neutral and hydrophobic amino acid, preferably G, T, V, A, P, M, D, or E, and more preferably V or G.
  • the fifth residue was C of the consensus sequence.
  • the sixth residue was a basic, or neutral and polar, or neutral and hydrophobic amino acid, preferably R, L, I, ,G, Q, T or S, and more preferably R.
  • the seventh residue was a basic, or neutral and polar, or neutral and hydrophobic amino acid, preferably R, G, N, P, W, or L, and more preferably R.
  • Residues 8 to 11 are the GWVG sequence of the conserved 12-mer.
  • Residue 12 was H, Q or N, preferably H, and the thirteen residue was the conserved C of the 12-mer.
  • the fourteenth residue was acidic, or basic or neutral and polar, preferably E, K, N, Q or S, more preferably N or K.
  • the fifteenth residue was an acidic, or neutral and polar, or neutral and hydrophobic amino acid, preferably D, E, Y, V or A, and more preferably D or E, most preferably D.
  • the sixteen residue was W of the consensus 12-mer and the seventeenth residue was a neutral and polar, or neutral and hydrophobic amino acid, preferably Q, G, L, I, P, F or M, and more preferably L.
  • the eighteenth residue was a basic, or neutral and polar, or neutral and
  • Residues 19 to 21 were the invariant DEY sequence consei ⁇ ed in all of the peptides due to the mutagenesis scheme used (see Example 7 below).
  • Residues 22 to 38 of the peptides shown in FIG. 4 also included subsets of the 20 naturally occurring ⁇ -amino acids.
  • residue 22 was A, T, N, S or E, preferably A
  • residue 23 was K, R, S, N or I, preferably K or R
  • residue 24 was P, I, N, T, Q, K, H, R or E, preferably N
  • residue 25 was P, T, R or H, preferably P
  • residue 26 was I, P, S, G, T, N, R or K, preferably R or G
  • residue 27 was L, S, N, T, Y, H or K, preferably Y
  • residue 28 was P, A, Q, G or S, preferably P
  • residue 29 was a branched chain amino acid, A, M, F, N, T, E or D, preferably V
  • residue 30 was P, A, V, T, Q,
  • residue 31 was P, N, Q, H, K, R or D, preferably P
  • residue 32 was I, G, S, N, T, Q or R, preferably G or S
  • residue 33 was L, V, f, T, Y, N, S, Q, K, H, E or D, with N, Y or K appearing most frequently.
  • Residue 34 was L, V, P, A, Y, S, K, R, H, D or E, preferably S
  • residue 35 was L, V, P, M, Y, N, S, R, or H, preferably one of the branched-chain amino acids, more preferably L.
  • Residue 36 was I, N, G, Q, S,
  • Residue 37 was P, L, T, G, S or R
  • residue 38 was P, L, A, T, S, H or R. Both residues 37 and 38 were preferably P.
  • These amino acid residues represent the sequences of the population of 22 EPOR binding clones whose amino acid sequences are shown in FIG. 4, which are representative of binding peptides. Therefore, although certain trends in
  • EPOR binding peptides may be discerned by examination of these sequences, particularly for preferred amino acids for some residues, they are not exclusive and EPOR binding peptides that retain the conserved 12-mer sequence with flanking sequences not shown by any of the sequences shown for binding clones in FIG. 4 are within the scope of this invention.
  • residue 23 in the binding clones was preferably the basic amino .acids K or R, representing a change from the neutral parental S residue, whereas K and R were not highly represented at residue 23 in the non-binding clones.
  • K and R were not highly represented at residue 23 in the non-binding clones.
  • a higher percentage of the non-binding clones retained the parental H at residue 31 than the binding clones.
  • Ex.ample 1 describes construction of a plasmid cloning vector, pcDNA3-IgGl, that encoded a portion of the chimeric IgG-EPOR protein used for probing phage display libraries.
  • This plasmid vector encoded a portion of the murine IgGl heavy chain and was designed to receive a polynucleotide cassette that encoded the ligand- binding domain of the EPOR.
  • Example 1 Expression Vector Encoding the C H2 .
  • C H3 and Hinge Domains of the Murine IgGl Heavy Chain A plasmid vector, pcDNA3, contains neomycin and ampicillin drug resistance (selectable marker) genes, ColEl, fl and SN40 origins of replication, and a multiple
  • the plasmid pcD ⁇ A3 was digested with Not I and Xho I restriction endonucleases and the digestion products separated electrophoretically on a 1% agarose gel using TBE buffer (89 mM Tris, pH 8.0, 89 mM boric acid, 2 mM EDTA (ethylene diamine tetraacetic acid)).
  • TBE buffer 89 mM Tris, pH 8.0, 89 mM boric acid, 2 mM EDTA (ethylene diamine tetraacetic acid)
  • the largest DNA fragment of the digest was gel-purified, ethanol precipitated, pelleted and dried briefly.
  • the dried pellet of purified DNA fragment was resuspended in TE buffer (10 mM Tris, pH 7.5, 1 mM EDTA) and stored at -20°C.
  • This linearized plasmid was used to receive a polynucleotide that encoded a portion of a murine immunoglobulin (
  • a polynucleotide encoding the constant region CH2, CH3 and hinge domains of the murine IgGl heavy chain was amplified from genomic DNA using a PCR protocol employing primers having the following sequences.
  • First strand primer was (SEQ ID NO:2):
  • SEQ ID NO:2 corresponds to a Not I restriction endonuclease cleavage site
  • the bolded underlined portion of SEQ ID NO:3 corresponds to an Xho I restriction endonuclease cleavage site.
  • Mouse genomic DNA was prepared from a lysate of frozen NIH3T3 cells using standard laboratory procedures. Briefly, cells (5xl0 5 ) were pelleted by centrifugation, washed with phosphate-buffered saline, resuspended in 100 ⁇ l of a hypotonic buffer (50 mM KC1, 10 mM Tris HC1 (pH 8.4), 1.5 mM MgCl 2 ) containing 0.5% (v/v) nonionic
  • the PCR reaction for amplifying the polynucleotide region encoding the CH2, CH3 and hinge domains of the murine IgGl heavy chain was prepared by combining the following reagents in a sterile 0.6 ml microfuge tube in the following order: 10 ⁇ l of 10X PCR Buffer H (100 mM Tris HC1 (pH 8.3), 500 mM KC1), 6 ⁇ l of 25 mM
  • Amplified DNA from the PCR reaction was gel purified by electrophoresis through a 1 % agarose gel in TBE. The band corresponding to the amplified DNA was excised from the gel and eluted in 40 ⁇ l of water.
  • the amplified IgGl gene fragment of about 1 kb was then digested with Not I and Xho I restriction endonucleases, and the digestion products electrophoresed on a 1% agarose/TBE gel.
  • the about 1 kb DNA fragment was again purified from the gel and eluted in 40 ⁇ l of water. The yield of the purified fragment was determined by measuring the optical density of the solution at
  • the Xho I and Not I digested IgGl PCR product was ligated into the Xho I and Not I digested pcDNA3 vector in a 20 ⁇ l ligation reaction containing about 100 ng each of the pcDNA3 vector and IgGl amplified DNA fragment, in 50 mM Tris-HCl (pH 7.8), 10 mM MgCl 2 , 10 mM dithiothreitol (DTT), 1 mM ATP, 25 ⁇ g/mL bovine serum albumin (BSA) and 1 unit of DNA ligase, incubated overnight at room temperature.
  • Not I and Xho I and resolved on a 1 % agarose TBE analytical gel to check for the presence of the polynucleotide segment encoding the murine IgGl constant and hinge regions.
  • Plasmid DNA from clones containing the Not I/Xho I insert was prepared for nucleic acid sequencing.
  • Nucleic acid sequencing of the Not I Xho I insert was performed using a dideoxy sequencing protocol. Sequencing reaction mixtures were run on a 4% acrylamide denaturing gels containing urea for 10 hours and the entire sequence of the fragment determined. After verifying that one of the clones, designated pcDNA3-IgGl
  • -17- (SEQ ID NO:4), contained an insert having the proper sequence (i.e., murine IgGl C m , C H3 , and hinge regions), a large-scale plasmid preparation was carried out.
  • the pcDNA3-IgGl expression vector was used to create a new expression plasmid encoding a chimeric EPOR protein useful for probing phage display or other combinatorial libraries for ligands, as described in Example 2. It will be appreciated by those skilled in the art that the pcDNA3-IgGl vector may be used as a recipient DNA for sequences encoding other peptides, thereby allowing one to easily create other chimeric proteins useful in similar types of probing assays. That is, the pcDNA3-IgGl vector is a general pu ⁇ ose vector for producing chimeric proteins that include a portion of the murine
  • the following example describes the methods used to construct a plasmid expression vector that encoded a chimeric protein having the C ⁇ , C H3 , and hinge regions of murine IgGl and the ligand-binding domain of the human EPOR.
  • the mixture was incubated for 1 hour at 42 °C, heat-treated at 95 °C for
  • PCR reactions were performed using the following primers.
  • the first strand primer was:
  • the first strand primer (SEQ ID NO:5) inco ⁇ orated into the amplification product an ATG translation start codon (shown underlined above) and a Bam HI
  • Example 1 but with the primers having the sequences of SEQ ID NO:5 .and SEQ ID NO:6, and yielded an amplified EPOR DNA fragment having the sequence of SEQ ID NO:7.
  • the amplified EPOR DNA fragment (i.e., the PCR product) and the pcDNA3- IgGl plasmid each were digested with Bam HI and Not I, and the large DNA fragments of each reaction were gel purified using methods as described in Example 1.
  • the purified EPOR DNA fragment and plasmid vector were then ligated, using ligation conditions substantially as described in Example 1, to yield the chimeric expression vector designated pcDNA3-IgGl-EPOR. This chimeric expression vector was transfected into competent E.
  • RNA transcribed from the vector-borne CMV promoter was translated within the transfected cells to yield a fusion protein having domains corresponding to murine IgGl and the extracellular domain (ECD) of the human EPOR.
  • Vector construction was confi ⁇ ned by diagnostic restriction digests and nucleic acid sequencing using standard methods. Large scale plasmid preparations were made from a transformed E. coli clone harboring the pcDNA3-IgGl-EPOR plasmid.
  • Example 3 Transfection of the pcDNA3-IgGl-EPOR Construct and Expression of the Chimeric Ig-EPOR Protein COS-7 cells were propagated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 4500 mg/L D-glucose, 584 mg/ml L-glutamine, and 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • proteins in the cell free supematants contained one moiety capable of binding to authentic EPO and another moiety capable of binding to an anti-IgG antibody. That is, transfectants containing the pcDNA3-IgGl-EPOR plasmid expressed a chimeric protein that included functional IgGl and EPOR regions.
  • Example 4 describes the ELISA methods used to confirm that the chimeric Ig- EPOR protein was secreted from cells transfected with the pcDNA3-IgGl-EPOR plasmid and describes the method used to demonstrate that the chimeric protein simultaneously could bind authentic EPO and a labeled anti-IgG antibody.
  • the chimeric IgG-EPOR protein was suitable as a probe for identifying EPOR ligands. That is, the chimeric Ig-EPOR exhibited specific binding to EPO, and also specifically bound an anti-murine IgG antibody.
  • the following example describes the methods used to purify the chimeric Ig- EPOR protein used to identify EPOR ligands.
  • ELISA ELISA and a known concentration of antibody, all according to standard laboratory procedures substantially as described in Example 4.
  • the integrity of the chimeric protein was determined by PAGE on 4% to 20% Tris-glycine pre-cast acrylamide gels and silver staining (Bio-Rad, Hercules CA), and immunoblotting using HRP labeled goat anti-murine IgGl to detect the Ig-EPOR protein, also according to standard laboratory procedures.
  • Example 6 describes the methods used to isolate recombinant phage that expressed fusion proteins capable of binding the chimeric Ig-EPOR probe.
  • M13 phage display libraries which expressed random peptides as a pIH fusion protein were screened independently using biopanning with the Ig-EPOR probe. These libraries, designated XC8, XC13, X38, XC43, were constructed using previously described procedures (McConnell et al., Molec. Diversity 1 : 165-176, 1996).
  • the XC8 and XC13 libraries displayed random amino acid sequences of 8 and 13 residues, respectively, flanked by cysteine residues.
  • the XC38 library expressed a fusion protein displaying 37 random amino acids surrounding an invariant alanine at position 22 in the peptide sequence.
  • the XC43 library displayed 43-mer peptides comprising 40 random amino acids suirounding an invariant Gly-Cys-Gly sequence at residues 21, 22 and 23, which allows the potential for a displayed peptide to form an intramolecular disulfide loop.
  • the phage libraries were screened for EPOR binding clones using the above- described chimeric Ig-EPOR protein as a probe using standard biopanning procedures.
  • Biopanning was performed using the chimeric receptor protein immobilized to
  • Beads were further treated by addition of the Fc fragment of murine IgG (10 ⁇ g/ml), for lhr at room temperature to block high affinity sites that could non-specifically bind the IgG domain of the chimeric probe.
  • the beads were washed three times in TBST (Tris buffered saline containing 0.1% (v/v) TWEEN " 20) and then suspended in 1 ml blocking medium (TBS SUPERBLOCK® ⁇ . Phage in 100 ⁇ l aliquots containing about 1 x 10'° plaque forming units (pfu) were added to the beads and the mixtures rotated gently for 2 hrs at room temperature.
  • phage were eluted with 300 ⁇ l of 50 mM glycine buffer (pH 2.2) for 10 min at room temperature. Eluted phage were immediately neutralized with 8 ⁇ l 2M Tris base that had not been pH-adjusted, mixed and stored at 4°C. Phage isolated by biopanning were amplified by mixing equal volumes of eluted phage and an
  • ERB 1 for "EPO receptor binder 1 "
  • ERB 1 for "EPO receptor binder 1 "
  • the ERB 1 clone included a polynucleotide having the sequence of SEQ ID NO: 8 fused to sequences encoding the pffl coat protein.
  • the amino acid sequence of the EPOR-binding peptide contained within the ERB 1 clone was: DFDVCRRGWVGHCKDWLSDEYASNPSYPVPHSYYLNPP (SEQ ID NO:9).
  • the amino acid sequence of the ERB 1 -encoded peptide was unrelated to the amino acid sequence of EPO, the ERB1 phage clone bound specifically to the Ig-EPOR protein.
  • a magnetic bead ELISA protocol was used to verify that the ERB1 phage clone bound to the chimeric Ig-EPOR probe by specific interactions.
  • the chimeric Ig-EPOR probe was bound to magnetic beads displaying rat anti-murine IgGl, substantially as described above.
  • Samples (100 ⁇ l) of 1:2 serial dilutions (PBS and 1% BSA) of a phage lysate were combined with the beads, and the phage allowed to bind the immobilized probe for 1 hr at room temperature.
  • Phage used in this procedure were ERB 1 and two negative control phage: HPKF, a clone that binds specifically to a commercially obtained anti-IL-8 mAb, and IL6-B26, a clone that binds specifically to a human IL-6 receptor.
  • HPKF HPKF
  • IL6-B26 a clone that binds specifically to a human IL-6 receptor.
  • the HRP- conjugated anti-M13 detection antibody was added to all samples and incubated (1 hr, room temperature) and then excess detection antibody was removed by washing.
  • the washed bead complexes (10 ⁇ l) were transferred to 96-well microtiter wells, developed using the T.MB peroxidase substrate, and the reaction measured spectrophotometrically, substantially as described above.
  • the chimeric Ig-EPOR probe described herein represented the binding characteristics of the EPOR and molecules that bound the chimeric probe would be expected to bind to EPOR.
  • an evolved library was created using saturation oligonucleotide doping mutagenesis of the original ERB 1 sequence and the evolved library was screened using the chimeric Ig-EPOR probe.
  • the following example describes the methods used to create and screen a phage display library of 38-mer peptides that were related to the fusion protein expressed by the ERB1 phage clone.
  • Example 7 Construction and Screening of an ERB 1 Evolved Library .An evolved library was constructed using procedures substantially as described previously (McConnell et al., 1996, Molecular Diversity 1:165). Briefly, we synthesized a collection of oligonucleotides such that codons corresponding to each amino acid position of the 38-mer ERB1 EPOR-binding peptide were independently substituted by random amino acids at a predetermined frequency, except for the DEY internal amino acid sequence near the center of the ERB 1 sequence. Using these procedures, codons representing substitutions of random amino acids at any single position in the 38-mer peptide, except those encoding the DEY tripeptide, were predicted to occur at a frequency of approximately 50%.
  • Polynucleotides were synthesized according to the doping scheme diagramed in FIG. 3. Briefly, two collections of redundant oligonucleotides, represented by the oligonucleotides of Fig. 3A (SEQ ID NO: 10 and SEQ ID NO: 11), were synthesized so that oligonucleotides of the two groups could be annealed to each other using short complementary sequences present at their 3' ends. These complementary sequences encode the internal D-E-Y amino acids of the ERB 1 peptide, thus maintaining this tripeptide in each of the clones of the resulting evolved library. The complementary strands of the population of annealed oligonucleotides were then synthesized by standard in vitro DNA extension methods to create a population of double-stranded
  • DNA fragments were cloned into an appropriate M13 vector (e.g., MSM1 of McConnell et al., supra) to produce a phage display library that expressed pin fusion proteins, where each fusion protein includes an N-terminal 38-mer
  • the redundant oligonucleotides were synthesized using 73% of the nucleotide identical to the nucleotide used in that position of the ERB1 oligonucleotide sequence and 9% of the other three nucleotides.
  • the nucleotide compositions that correspond to each base shown in lower case in the redundant oligonucleotide sequences are shown in FIG. 3B.
  • the third position of each codon (represented by "s" in .FIGS. 3A and 3B) was synthesized using an equimolar mixture of dGTP and dCTP.
  • This doping scheme produced nucleotide triplets encoding the original amino acid at each position of the ERB1 38-mer peptide approximately 50% of the time. The remaining 50% of the time, codons that did not encode the original amino acid were substituted by a random mixture of the other 19 amino acids.
  • ERB 1 evolved library was screened by biopanning substantially as described in Example 6 to identify clones that bound the chimeric Ig-EPOR probe.
  • Several random phage clones also were isolated from the evolved library, to represent non-binding clones.
  • approximately 88% of clones selected from the panned group were positive for EPOR binding, whereas all of the randomly selected clones from the unpanned group were negative for EPOR binding. This indicated that randomly selected clones in the evolved library were unlikely to bind the EPOR, whereas clones selected by the panning procedure exhibited EPOR binding at a very high frequency.
  • the screening methods described herein are useful for identifying and isolating phage clones that exhibit ligand functionality; in this case, clones capable ofbinding to EPOR.
  • a total of 48 binding (panned group) and 24 non-binding (unpanned group) clones were isolated from the evolved library. DNA sequences encoding the 38-mer regions of the fusion peptides these clones were determined by dideoxy sequencing using standard procedures. Among these 72 isolates, 34 unique sequences were found (21 sequences for the panned group and 13 sequences for the unpanned group). These 34 unique DNA sequences are disclosed in SEQ E NO: 13 to SEQ ID NO:46 and the predicted amino acid sequences encoded by these sequences are disclosed in SEQ ID
  • Nl to N13 SEQ ED NO:68 to SEQ ID NO:80
  • binding and non-binding clones all included the same pHI protein sequences, binding of the phage clones to the EPOR probe was not due to interaction between the probe and pIU sequences. Instead, binding of the phage clones to the Ig-EPOR probe must have been due to the presence of additional peptide sequences of the fusion protein (e.g., those shown for clones B 1 to B21 in Figure 4).
  • additional peptide sequences of the fusion protein e.g., those shown for clones B 1 to B21 in Figure 4.
  • peptides that included the 12-mer consensus sequence bound to the ligand binding domain of the human EPOR and peptides comprising the sequence of SEQ ID NO: 12 represented EPO peptide mimetics.
  • the optical density per phage at 50% ERB1 maximum response multiplied by lxlO 8 gave a value of 1.0 for ERB1, and represented the standard for measuring relative binding affinity of the Bl to B21 isolates.
  • the normalized relative affinity ("Rel. Aff.") for each clone is shown to the right of the clone's peptide sequence in FIG. 4. For each of the non-binding clones, the relative affinity is listed as ⁇ 0.1 because each of these was negative for EPOR binding.
  • Figures 5A - 5D graphically show the ELISA results of clones Bl to B2..
  • Example 8 Peptide Inhibition ELISA This example shows that synthetic peptides that include the consensus sequence of SEQ ED NO: 12 competed with EPO for EPOR binding. Because the consensus sequence was positioned on the amino-terminal side of the conserved DEY residues, peptides were synthesized based on the amino-terminal sequences of two exempl-ary clones that exhibited high affinity binding for the chimeric Ig-EPOR probe. The peptides were synthesized in the cyclic form and include sequences corresponding to the amino-terminal 18 amino acids of the B7 and B8 clones.
  • Synthetic peptides having the sequences DREGCRRGWVGQCKAWFN (SEQ ID NO:81), and DVEACGGGWVGHCNYWLR (SEQ ID NO:82) were synthesized in the cyclic (disulfide-bonded) form using standard procedures (Peninsula Laboratories, Belmont, CA).
  • the 18-mer peptide sequences were derived from the B7 and B8 clones, and the peptides are referred to herein as "ERB 1-7" (SEQ ID NO:81) and "ERB 1-8"
  • ERB 1-8 synthetic peptides competed with EPO for binding to the chimeric Ig-EPOR protein. Both peptides had IC 50 values of about 45 nM in the assay. This proved that synthetic 18-mer peptides derived from the sequences of the binding clones (see FIG.
  • ERB 1-7 were synthesized and tested to determine the relationship of peptide size and receptor binding activity. As described in the following example, a series of N-te ⁇ ninal truncations of ERB 1-7 were synthesized and tested in the EPO specific inhibition
  • 17-mer peptide 7N-1 consisting of REGCRRGWVGQCKAWFN (SEQ ID NO:83); 16-mer peptide 7N-2, consisting of EGCRRGWVGQCKAWFN (SEQ ID NO:84);
  • 15-mer peptide 7N-3 consisting of GCRRGWVGQCKAWFN (SEQ ID NO:85); and 14-mer peptide 7N-4, consisting of CRRGWVGQCKAWFN (SEQ ID NO:86).
  • 17-mer peptide showed a decreased IC 50 of 91 nM. Further truncation produced the 16- mer that had an IC 50 of about 3700 nM. Further truncations (i.e., to 15-mer and 14-mer peptides) abolished EPOR binding activity. Truncation of the ERB 1-7 peptide not only
  • TF-1 cell line is useful for detecting and quantitating EPO biological activity, although other EPO-responsive cells could be substituted.
  • the TF-1 cell line was established from a heparinized bone marrow aspirate isolated from a 35-year-old male exhibiting pancytopenia (Kitamura et al., 1989, J. Physiol. 140:323).
  • TF-1 cells were propagated in RPMI 1640 containing 10% BCS and 5 ng/ml GM-CSF. After wasliing twice with PBS to remove residual GM-CSF, 5,000 cells were placed in each well of a 96-well microtiter plate in the presence of 0.5 units EPO/ml and cultured for three days under standard conditions (Kitamura et al., 1989, Blood 73:375- 380).
  • Proliferation in response to EPO was determined using a standard XXT assay (assay based on sodium 3'-(-l-(phenylaminocarbonyl)-3,4-tetrazolium)-bis (4-methoxy- 6-notro) benzene sulfonic acid hydrate; Promega, Madison WI). Similar procedures were followed using the ERB 1-7 (SEQ ID NO:81) or ERB 1-8 synthetic peptide (SEQ ID NO: 82 ) instead of EPO. Also tested as a negative control was an EL-6 peptide having the sequence GGAFCEAVGCGPDRNFYGG (SEQ ID NO:87). Peptide concentrations were tested over a 1, 000-fold concentration range of 0.02 to20 ⁇ M.
  • FIGS. 8 A and 8B show that the synthetic peptides which bound the EPOR also exhibited EPO-like pharmacologic activity in a biological assay.
  • GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC 2100
  • CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC 2340
  • GTTTTTCGCC CTTTGACGTT GGAGTCCACG TTCTTTAATA GTGGACTCTT GTTCCAAACT 2520
  • GACTTCGAAG ACTGCCAGGG TGGTTGGGTT GGTCACTGCA ACGACTGGCT GGGTGACGAA 60 TACGCTCGTC ACCCGCGTTA CGGTGCTACC CAGACCCTGT CTGTTAACCG TCAC 114
  • GACTTCGAAG ACTGCCAGGG TGGTTGGGTT GGTCACTGCA ACGACTGGCT GGGTGACGAA 60 TACGCTCGTC ACCCGCGTTA CGGTGCTACC CAGACCCTGT CTGTTAACCG TCAC 114
  • GAAGGTGAAG TTTGCCTGCC GGGTTGGGTT GGTCACTGCA AATACTGGCT GATGGACGAA 60 TACGCTAACA TCCCGCGTAA CCCGACCCCG CGTTCTAACG AACTGAAACC GCCG 114
  • GGTCTGAACA TCTGCCGTCC GGGTTGGGTT GGTCACTGCA ACGACTCTCT GCGTGACGAA 60 TACGCTACCA ACCCGCGTAC CCCGGGTCCG CTGTCTTACA ACCTGCAGCC GACC 114

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Abstract

La présente invention concerne des clones isolés à partir d'une bibliothèque d'affichage de phages se liant à une sonde de récepteur de l'érythropoietine (EPO). L'invention concerne également des peptides codés par des séquences desdits clones se liant au récepteur de l'EPO. Finalement l'invention concerne une séquence consensus d'acides aminés de 12 mer, CXXGWVGXCXXW (où X désigne un parmi plusieurs acides aminés), commune aux peptides se liant au récepteur de l'EPO, mais sans aucun lien avec la structure primaire de l'EPO.
PCT/US1999/005842 1998-03-20 1999-03-17 Ligands peptidiques pour recepteur d'erythropoietine Ceased WO1999047151A1 (fr)

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US7442778B2 (en) 2004-09-24 2008-10-28 Amgen Inc. Modified Fc molecules
US7488590B2 (en) 1998-10-23 2009-02-10 Amgen Inc. Modified peptides as therapeutic agents
US8143380B2 (en) 2004-07-08 2012-03-27 Amgen Inc. Therapeutic peptides
EP2594286A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
US9145450B2 (en) 1998-10-23 2015-09-29 Amgen Inc. Thrombopoietic compounds
CN110361535A (zh) * 2018-04-09 2019-10-22 宁夏医科大学总医院 检测促红细胞生成素受体的放射性探针、制备方法及应用

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MCCONNELL S. J., ET AL.: "ISOLATION OF ERYTHROPOIETIN RECEPTOR AGONIST PEPTIDES USING EVOLVED PHAGE LIBRARIES.", BIOLOGICAL CHEMISTRY, WALTER DE GRUYTER GMBH & CO., BERLIN, DE, vol. 379., 1 October 1998 (1998-10-01), BERLIN, DE, pages 1279 - 1286., XP002920440, ISSN: 1431-6730 *
WRIGHTON N. C., ET AL.: "SMALL PEPTIDES AS POTENT MIMETICS OF THE PROTEIN HORMONE ERYTHROPOIETIN.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 273., 26 July 1996 (1996-07-26), US, pages 458 - 463., XP002021036, ISSN: 0036-8075, DOI: 10.1126/science.273.5274.458 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7488590B2 (en) 1998-10-23 2009-02-10 Amgen Inc. Modified peptides as therapeutic agents
US9534032B2 (en) 1998-10-23 2017-01-03 Amgen Inc. Thrombopoietic compounds
US9145450B2 (en) 1998-10-23 2015-09-29 Amgen Inc. Thrombopoietic compounds
US8143380B2 (en) 2004-07-08 2012-03-27 Amgen Inc. Therapeutic peptides
US7442778B2 (en) 2004-09-24 2008-10-28 Amgen Inc. Modified Fc molecules
US9114175B2 (en) 2005-08-12 2015-08-25 Amgen Inc. Modified Fc molecules
US11266744B2 (en) 2005-08-12 2022-03-08 Amgen Inc. Modified Fc molecules
US10188740B2 (en) 2005-08-12 2019-01-29 Amgen Inc. Modified Fc molecules
EP2594287A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
EP2594288A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
US9283260B2 (en) 2006-04-21 2016-03-15 Amgen Inc. Lyophilized therapeutic peptibody formulations
EP2594284A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
US10166189B2 (en) 2006-04-21 2019-01-01 Amgen Inc. Lyophilized therapeutic peptibody formulations
EP2594285A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
EP2594286A1 (fr) 2006-04-21 2013-05-22 Amgen Inc. Formulations peptide-anticorps thérapeutiques lyophilisées
CN110361535A (zh) * 2018-04-09 2019-10-22 宁夏医科大学总医院 检测促红细胞生成素受体的放射性探针、制备方法及应用

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