US20040071712A1 - Modified peptides as therapeutic agents - Google Patents

Modified peptides as therapeutic agents Download PDF

Info

Publication number
US20040071712A1
US20040071712A1 US10/645,761 US64576103A US2004071712A1 US 20040071712 A1 US20040071712 A1 US 20040071712A1 US 64576103 A US64576103 A US 64576103A US 2004071712 A1 US2004071712 A1 US 2004071712A1
Authority
US
United States
Prior art keywords
gt
lt
feature
leu
pro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/645,761
Inventor
Ulrich Feige
Chuan-Fa Liu
Janet Cheetham
Thomas Boone
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amgen Inc
Original Assignee
Amgen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
Priority to US10537198P priority Critical
Priority to US09/428,082 priority patent/US6660843B1/en
Application filed by Amgen Inc filed Critical Amgen Inc
Priority to US10/645,761 priority patent/US20040071712A1/en
Publication of US20040071712A1 publication Critical patent/US20040071712A1/en
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=26802505&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20040071712(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application status is Abandoned legal-status Critical

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489Metalloendopeptidases (3.4.24)
    • C12N9/6491Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/505Erythropoietin [EPO]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/524Thrombopoietin, i.e. C-MPL ligand
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/525Tumour necrosis factor [TNF]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/545IL-1
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81Protease inhibitors
    • C07K14/8107Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8146Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention concerns fusion of Fc domains with biologically active peptides and a process for preparing pharmaceutical agents using biologically active peptides. In this invention, pharmacologically active compounds are prepared by a process comprising:
a) selecting at least one peptide that modulates the activity of a protein of interest; and
b) preparing a pharmacologic agent comprising an Fc domain covalently linked to at least one amino acid of the selected peptide.
Linkage to the vehicle increases the half-life of the peptide, which otherwise would be quickly degraded in vivo. The preferred vehicle is an Fc domain. The peptide is preferably selected by phage display, E. coli display, ribosome display, RNA-peptide screening, or chemical-peptide screening.

Description

    BACKGROUND OF THE INVENTION
  • Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemical modification. Such modifications can protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Protein modifications may also increase the therapeutic protein's stability, circulation time, and biological activity. A review article describing protein modification and fusion proteins is Francis (1992), [0001] Focus on Growth Factors 3:4-10 (Mediscript, London), which is hereby incorporated by reference.
  • One useful modification is combination with the “Fc” domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and a constant domain known as “Fc”, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), [0002] Nature 337: 525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusions known in the art.
    TABLE 1
    Fc fusion with therapeutic proteins
    Fusion Therapeutic
    Form of Fc partner implications Reference
    IgG1 N-terminus of Hodgkin's disease; U.S. Pat. No.
    CD30-L anaplastic lymphoma; T- 5,480,981
    cell leukemia
    Murine Fcγ2a IL-10 anti-inflammatory; Zheng et al. (1995), J. Immunol.
    transplant rejection 154: 5590-600
    IgG1 TNF receptor septic shock Fisher et al. (1996), N. Engl.
    J. Med. 334: 1697-1702;
    Van Zee, K. et al.
    (1996), J. Immunol. 156:
    2221-30
    IgG, IgA, TNF receptor inflammation, U.S. Pat. No. 5,808,029,
    IgM, or IgE autoimmune disorders issued Sep. 15, 1998
    (excluding
    the first
    domain)
    IgG1 CD4 receptor AIDS Capon et al. (1989),
    Nature 337: 525-31
    IgG1, N-terminus anti-cancer, antiviral Harvill et al. (1995),
    IgG3 of IL-2 Immunotech. 1: 95-105
    IgG1 C-terminus of osteoarthritis; WO 97/23614, published
    OPG bone density Jul. 3, 1997
    IgG1 N-terminus of anti-obesity PCT/US 97/23183, filed
    leptin Dec. 11, 1997
    Human Ig CTLA-4 autoimmune disorders Linsley (1991), J. Exp.
    Cγ1 Med. 174: 561-9
  • A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface contribute to most of the binding energy. Clackson et al. (1995), [0003] Science 267: 383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only “peptide” length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivity of the large protein ligand (“peptide agonists”) or, through competitive binding, inhibit the bioactivity of the large protein ligand (“peptide antagonists”).
  • Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), [0004] Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of which is incorporated by reference). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, e.g., Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24.
  • Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, e.g., Takasaki et al. (1997), [0005] Nature Biotech. 15: 1266-70. These analytical methods may also be used to investigate the interaction between a receptor protein and peptides selected by phage display, which may suggest further modification of the peptides to increase binding affinity.
  • Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in [0006] E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as “E. coli display.” In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as “ribosome display.” Other methods employ chemical linkage of peptides to RNA; see, for example, Roberts & Szostak (1997), Proc. Natl. Acad. Sci. USA, 94:12297-303. Hereinafter, this and related methods are collectively referred to as “RNA-peptide screening.” Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as “chemical-peptide screening.” Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.
  • Conceptually, one may discover peptide mimetics of any protein using phage display and the other methods mentioned above. These methods have been used for epitope mapping, for identification of critical amino acids in protein-protein interactions, and as leads for the discovery of new therapeutic agents. E.g., Cortese et al. (1996), [0007] Curr. Opin. Biotech. 7: 616-21. Peptide libraries are now being used most often in immunological studies, such as epitope mapping. Kreeger (1996), The Scientist 10(13): 19-20.
  • Of particular interest here is use of peptide libraries and other techniques in the discovery of pharmacologically active peptides. A number of such peptides identified in the art are summarized in Table 2. The peptides are described in the listed publications, each of which is hereby incorporated by reference. The pharmacologic activity of the peptides is described, and in many instances is followed by a shorthand term therefor in parentheses. Some of these peptides have been modified (e.g., to form C-terminally cross-linked dimers). Typically, peptide libraries were screened for binding to a receptor for a pharmacologically active protein (e.g., EPO receptor). In at least one instance (CTLA4), the peptide library was screened for binding to a monclonal antibody. [0008]
    TABLE 2
    Pharmacologically active peptides
    Binding
    partner/
    Form of protein of Pharmacologic
    peptide interesta activity Reference
    intrapeptide EPO receptor EPO-mimetic Wrighton et al. (1996),
    disulfide- Science 273: 458-63;
    bonded U.S. Pat. No. 5,773,569,
    issued Jun. 30, 1998 to
    Wrighton et al.
    C-terminally EPO receptor EPO-mimetic Livnah et al. (1996),
    cross-linked Science 273: 464-71;
    dimer Wrighton et al. (1997),
    Nature Biotechnology 15:
    1261-5; International
    patent application WO
    96/40772, published
    Dec. 19, 1996
    linear EPO receptor EPO-mimetic Naranda et al. (1999),
    Proc. Natl. Acad. Sci.
    USA, 96: 7569-74
    linear c-Mpl TPO-mimetic Cwirla et al. (1997)
    Science 276: 1696-9;
    U.S. Pat. No. 5,869,451,
    issued Feb. 9, 1999; U.S.
    Pat. No. 5,932,946,
    issued Aug. 3, 1999
    C-terminally c-Mpl TPO-mimetic Cwirla et al. (1997),
    cross-linked Science 276: 1696-9
    dimer
    disulfide- stimulation of Paukovits et al. (1984),
    linked dimer hematopoiesis Hoppe-Seylers Z.
    (“G-CSF-mimetic”) Physiol. Chem. 365: 303-11;
    Laerum et al. (1988),
    Exp. Hemat. 16: 274-80
    alkylene- G-CSF-mimetic Bhatnagar et al. (1996),
    linked dimer J. Med. Chem. 39: 3814-9;
    Cuthbertson et al.
    (1997), J. Med. Chem.
    40: 2876-82; King et al.
    (1991), Exp. Hematol.
    19: 481; King et al.
    (1995), Blood 86 (Suppl.
    1): 309a
    linear IL-1 receptor inflammatory and U.S Pat. No. 5,608,035;
    autoimmune diseases U.S. Pat. No. 5,786,331;
    (“IL-1 antagonist” or U.S. Pat. No. 5,880,096;
    “IL-1ra-mimetic”) Yanofsky et al. (1996),
    Proc. Natl. Acad. Sci. 93:
    7381-6; Akeson et al.
    (1996), J. Biol. Chem.
    271: 30517-23;
    Wiekzorek et al. (1997),
    Pol. J. Pharmacol. 49:
    107-17; Yanofsky (1996),
    PNAs, 93: 7381-7386.
    linear Facteur stimulation of Inagaki-Ohara et al.
    thymique lymphocytes (1996), Cellular Immunol.
    serique (FTS) (“FTS-mimetic”) 171: 30-40; Yoshida
    (1984), Int. J. Immunopharmacol,
    6: 141-6.
    intrapeptide CTLA4 MAb CTLA4-mimetic Fukumoto et al. (1998),
    disulfide Nature Biotech. 16: 267-70
    bonded
    exocyclic TNF-α receptor TNF-α antagonist Takasaki et al. (1997),
    Nature Biotech. 15: 1266-70;
    WO 98/53842,
    published Dec. 3, 1998
    linear TNF-α receptor TNF-α antagonist Chirinos-Rojas ( ), J. Imm.,
    5621-5626.
    intrapeptide C3b inhibition of complement Sahu et al. (1996), J. Immunol.
    disulfide activation; autoimmune 157: 884-91;
    bonded diseases Morikis et al. (1998),
    (“C3b-antagonist”) Protein Sci. 7: 619-27
    linear vinculin cell adhesion processes - Adey et al. (1997),
    cell growth, differentiation, Biochem. J. 324: 523-8
    wound healing, tumor
    metastasis (“vinculin
    binding”)
    linear C4 binding anti-thrombotic Linse et al. (1997), J. Biol. Chem.
    protein (C4BP) 272: 14658-65
    linear urokinase processes associated with Goodson et al. (1994),
    receptor urokinase interaction with Proc. Natl. Acad. Sci. 91:
    its receptor (e.g., 7129-33; International
    angiogenesis, tumor cell application WO
    invasion and metastasis); 97/35969, published
    (“UKR antagonist”) Oct. 2, 1997
    linear Mdm2, Hdm2 Inhibition of inactivation of Picksley et al. (1994),
    p53 mediated by Mdm2 or Oncogene 9: 2523-9;
    hdm2; anti-tumor Bottger et al. (1997) J. Mol. Biol.
    (“Mdm/hdm antagonist”) 269: 744-56;
    Bottger et al. (1996),
    Oncogene 13: 2141-7
    linear p21WAF1 anti-tumor by mimicking Ball et al. (1997), Curr.
    the activity of p21WAF1 Biol. 7: 71-80
    linear farnesyl anti-cancer by preventing Gibbs et al. (1994), Cell
    transferase activation of ras oncogene 77: 175-178
    linear Ras effector anti-cancer by inhibiting Moodie et al. (1994),
    domain biological function of the Trends Genet 10: 44-48
    ras oncogene Rodriguez et al. (1994),
    Nature 370: 527-532
    linear SH2/SH3 anti-cancer by inhibiting Pawson et al (1993),
    domains tumor growth with Curr. Biol. 3: 434-432
    activated tyrosine kinases Yu et al. (1994), Cell
    76: 933-945
    linear p16INK4 anti-cancer by mimicking F$$hraeus et al. (1996),
    activity of p16; e.g., Curr. Biol. 6: 84-91
    inhibiting cyclin D-Cdk
    complex (“p16-mimetic”)
    linear Src, Lyn inhibition of Mast cell Stauffer et al. (1997),
    activation, IgE-related Biochem. 36: 9388-94
    conditions, type I
    hypersensitivity (“Mast
    cell antagonist”)
    linear Mast cell treatment of inflammatory International application
    protease disorders mediated by WO 98/33812, published
    release of tryptase-6 Aug. 6, 1998
    (“Mast cell protease
    inhibitors”)
    linear SH3 domains treatment of SH3- Rickles et al. (1994),
    mediated disease states EMBO J. 13: 5598-5604;
    (“SH3 antagonist”) Sparks et al. (1994), J. Biol. Chem.
    269: 23853-6;
    Sparks et al. (1996),
    Proc. Natl. Acad. Sci. 93:
    1540-4
    linear HBV core treatment of HBV viral Dyson & Muray (1995),
    antigen (HBcAg) infections (“anti-HBV”) Proc. Natl. Acad. Sci. 92:
    2194-8
    linear selectins neutrophil adhesion; Martens et al. (1995), J. Biol. Chem.
    inflammatory diseases 270: 21129-36;
    (“selectin antagonist”) European patent
    application EP 0 714
    912, published Jun. 5, 1996
    linear, calmodulin calmodulin antagonist Pierce et al. (1995),
    cyclized Molec. Diversity 1: 259-65
    Dedman et al.
    (1993), J. Biol. Chem.
    268: 23025-30; Adey & Kay
    (1996), Gene 169:
    133-4
    linear, integrins tumor-homing; treatment International applications
    cyclized- for conditions related to WO 95/14714, published
    integrin-mediated cellular Jun. 1, 1995; WO
    events, including platelet 97/08203, published
    aggregation, thrombosis, Mar. 6, 1997; WO
    wound healing, 98/10795, published
    osteoporosis, tissue Mar. 19, 1998; WO
    repair, angiogenesis (e.g., 99/24462, published May
    for treatment of cancer), 20, 1999; Kraft et al.
    and tumor invasion (1999), J. Biol. Chem.
    (“integrin-binding”) 274: 1979-1985
    cyclic, linear fibronectin and treatment of inflammatory WO 98/09985,
    extracellular and autoimmune published Mar. 12, 1998
    matrix conditions
    components of
    T cells and
    macrophages
    linear somatostatin treatment or prevention of European patent
    and cortistatin hormone-producing application 0 911 393,
    tumors, acromegaly, published Apr. 8, 1999
    giantism, dementia,
    gastric ulcer, tumor
    growth, inhibition of
    hormone secretion,
    modulation of sleep or
    neural activity
    linear bacterial antibiotic; septic shock; U.S. Pat. No. 5,877,151,
    lipopolysaccharide disorders modulatable by issued Mar. 2, 1999
    CAP37
    linear or pardaxin, antipathogenic WO 97/31019, published
    cyclic, mellitin Aug. 28, 1997
    including D-
    amino acids
    linear, cyclic VIP impotence, WO 97/40070, published
    neurodegenerative Oct. 30, 1997
    disorders
    linear CTLs cancer EP 0 770 624, published
    May 2, 1997
    linear THF-gamma2 Burnstein (1988),
    Biochem., 27: 4066-71.
    linear Amylin Cooper (1987), Proc.
    Natl. Acad. Sci.,
    84: 8628-32.
    linear Adrenomedullin Kitamura (1993), BBRC,
    192: 553-60.
    cyclic, linear VEGF anti-angiogenic; cancer, Fairbrother (1998),
    rheumatoid arthritis, Biochem., 37: 17754-17764.
    diabetic retinopathy,
    psoriasis (“VEGF
    antagonist”)
    cyclic MMP inflammation and Koivunen (1999), Nature
    autoimmune disorders; Biotech., 17: 768-774.
    tumor growth
    (“MMP inhibitor”)
    HGH fragment U.S. Pat. No. 5,869,452
    Echistatin inhibition of platelet Gan (1988), J. Biol. Chem.,
    aggregation 263: 19827-32.
    linear SLE SLE WO 96/30057, published
    autoantibody Oct. 3, 1996
    GD1alpha suppression of tumor Ishikawa et al. (1998),
    metastasis FEBS Lett. 441 (1): 20-4
    antiphospholipid endothelial cell activation, Blank et al. (1999), Proc.
    beta-2- antiphospholipid Natl. Acad. Sci. USA 96:
    glycoprotein-i syndrome (APS), 5164-8
    (β2GPI) thromboembolic
    antibodies phenomena,
    thrombocytopenia, and
    recurrent fetal loss
    linear T Cell Receptor diabetes WO 96/11214, published
    beta chain Apr. 18, 1996
  • Peptides identified by peptide library screening have been regarded as “leads” in development of therapeutic agents rather than as therapeutic agents themselves. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticuloendothelial system, or proteolytic degradation. Francis (1992), [0009] Focus on Growth Factors 3: 4-11. As a result, the art presently uses the identified peptides to validate drug targets or as scaffolds for design of organic compounds that might not have been as easily or as quickly identified through chemical library screening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24; Kay et al. (1998), Drug Disc. Today 3: 370-8. The art would benefit from a process by which such peptides could more readily yield therapeutic agents.
  • SUMMARY OF THE INVENTION
  • The present invention concerns a process by which the in vivo half-life of one or more biologically active peptides is increased by fusion with a vehicle. In this invention, pharmacologically active compounds are prepared by a process comprising: [0010]
  • a) selecting at least one peptide that modulates the activity of a protein of interest; and [0011]
  • b) preparing a pharmacologic agent comprising at least one vehicle covalently linked to at least one amino acid sequence of the selected peptide. [0012]
  • The preferred vehicle is an Fc domain. The peptides screened in step (a) are preferably expressed in a phage display library. The vehicle and the peptide may be linked through the N- or C-terminus of the peptide or the vehicle, as described further below. Derivatives of the above compounds (described below) are also encompassed by this invention. [0013]
  • The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable. [0014]
  • The primary use contemplated is as therapeutic or prophylactic agents. The vehicle-linked peptide may have activity comparable to—or even greater than—the natural ligand mimicked by the peptide. In addition, certain natural ligand-based therapeutic agents might induce antibodies against the patient's own endogenous ligand; the vehicle-linked peptide avoids this pitfall by having little or typically no sequence identity with the natural ligand. [0015]
  • Although mostly contemplated as therapeutic agents, compounds of this invention may also be useful in screening for such agents. For example, one could use an Fc-peptide (e.g., Fc-SH2 domain peptide) in an assay employing anti-Fc coated plates. The vehicle, especially Fc, may make insoluble peptides soluble and thus useful in a number of assays. [0016]
  • The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the instant invention. [0017]
  • Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.[0018]
  • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows a schematic representation of an exemplary process of the invention. In this preferred process, the vehicle is an Fc domain, which is linked to the peptide covalently by expression from a DNA construct encoding both the Fc domain and the peptide. As noted in FIG. 1, the Fc domains spontaneously form a dimer in this process. [0019]
  • FIG. 2 shows exemplary Fc dimers that may be derived from an IgG1 antibody. “Fc” in the figure represents any of the Fc variants within the meaning of “Fc domain” herein. “X[0020] 1” and “X2” represent peptides or linker-peptide combinations as defined hereinafter. The specific dimers are as follows:
  • A, D: Single disulfide-bonded dimers. IgG1 antibodies typically have two disulfide bonds at the hinge region between the constant and variable domains. The Fc domain in FIGS. 2A and 2D may be formed by truncation between the two disulfide bond sites or by substitution of a cysteinyl residue with an unreactive residue (e.g., alanyl). In FIG. 2A, the Fc domain is linked at the amino terminus of the peptides; in [0021] 2D, at the carboxyl terminus.
  • B, E: Doubly disulfide-bonded dimers. This Fc domain may be formed by truncation of the parent antibody to retain both cysteinyl residues in the Fc domain chains or by expression from a construct including a sequence encoding such an Fc domain. In FIG. 2B, the Fc domain is linked at the amino terminus of the peptides; in [0022] 2E, at the carboxyl terminus.
  • C, F: Noncovalent dimers. This Fc domain may be formed by elimination of the cysteinyl residues by either truncation or substitution. One may desire to eliminate the cysteinyl residues to avoid impurities formed by reaction of the cysteinyl residue with cysteinyl residues of other proteins present in the host cell. The noncovalent bonding or the Fc domains is sufficient to hold together the dimer. [0023]
  • Other dimers may be formed by using Fc domains derived from different types of antibodies (e.g., IgG2, IgM). [0024]
  • FIG. 3 shows the structure of preferred compounds of the invention that feature tandem repeats of the pharmacologically active peptide. FIG. 3A shows a single chain molecule and may also represent the DNA construct for the molecule. FIG. 3B shows a dimer in which the linker-peptide portion is present on only one chain of the dimer. FIG. 3C shows a dimer having the peptide portion on both chains. The dimer of FIG. 3C will form spontaneously in certain host cells upon expression of a DNA construct encoding the single chain shown in FIG. 3A. In other host cells, the cells could be placed in conditions favoring formation of dimers or the dimers can be formed in vitro. [0025]
  • FIG. 4 shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that may be used in this invention. [0026]
  • FIG. 5 shows a synthetic scheme for the preparation of PEGylated peptide 19 (SEQ ID NO: 3). [0027]
  • FIG. 6 shows a synthetic scheme for the preparation of PEGylated peptide 20 (SEQ ID NO: 4). [0028]
  • FIG. 7 shows the nucleotide and amino acid sequences (SEQ ID NOS: 5 and 6, respectively) of the molecule identified as “Fc-TMP” in Example 2 hereinafter. [0029]
  • FIG. 8 shows the nucleotide and amino acid sequences (SEQ. ID. NOS: 7 and 8, respectively) of the molecule identified as “Fc-TMP-TMP” in Example 2 hereinafter. [0030]
  • FIG. 9 shows the nucleotide and amino acid sequences (SEQ. ID. NOS: 9 and 10, respectively) of the molecule identified as “TMP-TMP-Fc” in Example 2 hereinafter. [0031]
  • FIG. 10 shows the nucleotide and amino acid sequences (SEQ. ID. NOS: 11 and 12, respectively) of the molecule identified as “TMP-Fc” in Example 2 hereinafter. [0032]
  • FIG. 11 shows the number of platelets generated in vivo in normal female BDF1 mice treated with one 100 μg/kg bolus injection of various compounds, with the terms defined as follows. [0033]
  • PEG-MGDF: 20 kD average molecular weight PEG attached by reductive amination to the N-terminal amino group of amino acids 1-163 of native human TPO, which is expressed in [0034] E. coli (so that it is not glycosylated);
  • TMP: the TPO-mimetic peptide having the amino acid sequence IEGPTLRQWLAARA (SEQ ID NO: 13); TMP-TMP: the TPO-mimetic peptide having the amino acid sequence IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ ID NO: 14); [0035]
  • PEG-TMP-TMP: the peptide of SEQ ID NO: 14, wherein the PEG group is a 5 kD average molecular weight PEG attached as shown in FIG. 6; [0036]
  • Fc-TMP-TMP: the compound of SEQ ID NO: 8 (FIG. 8) dimerized with an identical second monomer (i.e., Cys residues 7 and 10 are bound to the corresponding Cys residues in the second monomer to form a dimer, as shown in FIG. 2); and [0037]
  • TMP-TMP-Fc is the compound of SEQ ID NO: 10 (FIG. 9) dimerized in the same way as TMP-TMP-Fc except that the Fc domain is attached at the C-terminal end rather than the N-terminal end of the TMP-TMP peptide. [0038]
  • FIG. 12 shows the number of platelets generated in vivo in normal BDF1 mice treated with various compounds delivered via implanted osmotic pumps over a 7-day period. The compounds are as defined for FIG. 7. [0039]
  • FIG. 13 shows the nucleotide and amino acid sequences (SEQ. ID. NOS: 15 and 16, respectively) of the molecule identified as “Fc-EMP” in Example 3 hereinafter. [0040]
  • FIG. 14 shows the nucleotide and amino acid sequences (SEQ ID NOS: 17 and 18, respectively) of the molecule identified as “EMP-Fc” in Example 3 hereinafter. [0041]
  • FIG. 15 shows the nucleotide and amino acid sequences (SEQ ID NOS:19 and 20, respectively) of the molecule identified as “EMP-EMP-Fc” in Example 3 hereinafter. [0042]
  • FIG. 16 shows the nucleotide and amino acid sequences (SEQ ID NOS: 21 and 22, respectively) of the molecule identified as “Fc-EMP-EMP” in Example 3 hereinafter. [0043]
  • FIGS. 17A and 17B show the DNA sequence (SEQ ID NO: 23) inserted into pCFM1656 between the unique AatII (position #4364 in pCFM1656) and SacII (position #4585 in pCFM1656) restriction sites to form expression plasmid pAMG21 (ATCC accession no. 98113). [0044]
  • FIG. 18A shows the hemoglobin, red blood cells, and hematocrit generated in vivo in normal female BDF1 mice treated with one 100 μg/kg bolus injection of various compounds. FIG. 18B shows the same results with mice treated with 100 μg/kg per day delivered the same dose by 7-day micro-osmotic pump with the EMPs delivered at 100 μg/kg, rhEPO at 30 U/mouse. (In both experiments, neutrophils, lymphocytes, and platelets were unaffected.) In these figures, the terms are defined as follows. [0045]
  • Fc-EMP: the compound of SEQ ID NO: 16 (FIG. 13) dimerized with an identical second monomer (i.e., Cys residues 7 and 10 are bound to the corresponding Cys residues in the second monomer to form a dimer, as shown in FIG. 2); [0046]
  • EMP-Fc: the compound of SEQ ID NO: 18 (FIG. 14) dimerized in the same way as Fc-EMP except that the Fc domain is attached at the C-terminal end rather than the N-terminal end of the EMP peptide. [0047]
  • “EMP-EMP-Fc” refers to a tandem repeat of the same peptide (SEQ ID NO: 20) attached to the same Fc domain by the carboxyl terminus of the peptides. “Fc-EMP-EMP” refers to the same tandem repeat of the peptide but with the same Fc domain attached at the amino terminus of the tandem repeat. All molecules are expressed in [0048] E. coli and so are not glycosylated.
  • FIGS. 19A and 19B show the nucleotide and amino acid sequences (SEQ ID NOS: 1055 and 1056) of the Fc-TNF-α inhibitor fusion molecule described in Example 4 hereinafter. [0049]
  • FIGS. 20A and 20B show the nucleotide and amino acid sequences (SEQ ID NOS: 1057 and 1058) of the TNF-α inhibitor-Fc fusion molecule described in Example 4 hereinafter. [0050]
  • FIGS. 21A and 21B show the nucleotide and amino acid sequences (SEQ ID NOS: 1059 and 1060) of the Fc-IL-1 antagonist fusion molecule described in Example 5 hereinafter. [0051]
  • FIGS. 22A and 22B show the nucleotide and amino acid sequences (SEQ ID NOS: 1061 and 1062) of the IL-1 antagonist-Fc fusion molecule described in Example 5 hereinafter. [0052]
  • FIGS. 23A, 23B, and [0053] 23C show the nucleotide and amino acid sequences (SEQ ID NOS: 1063 and 1064) of the Fc-VEGF antagonist fusion molecule described in Example 6 hereinafter.
  • FIGS. 24A and 24B show the nucleotide and amino acid sequences (SEQ ID NOS: 1065 and 1066) of the VEGF antagonist-Fc fusion molecule described in Example 6 hereinafter. [0054]
  • FIGS. 25A and 25B show the nucleotide and amino acid sequences (SEQ ID NOS: 1067 and 1068) of the Fc-MMP inhibitor fusion molecule described in Example 7 hereinafter. [0055]
  • FIGS. 26A and 26B show the nucleotide and amino acid sequences (SEQ ID NOS: 1069 and 1070) of the MMP inhibitor-Fc fusion molecule described in Example 7 hereinafter.[0056]
  • DETAILED DESCRIPTION OF THE INVENTION
  • Definition of Terms [0057]
  • The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. [0058]
  • The term “comprising” means that a compound may include additional amino acids on either or both of the N- or C-termini of the given sequence. Of course, these additional amino acids should not significantly interfere with the activity of the compound. [0059]
  • The term “vehicle” refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein. Exemplary vehicles include an Fc domain (which is preferred) as well as a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); a branched-chain polymer (see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published Oct. 28, 1993); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor. Vehicles are further described hereinafter. [0060]
  • The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), [0061] Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.
  • The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published Sep. 25, 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference. Thus, the term “Fc variant” comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter. [0062]
  • The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means. [0063]
  • The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. Multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc. [0064]
  • The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in FIG. 2. [0065]
  • The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR[0066] 1, NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R1 and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R2 or —NR3R4 wherein R2, R3 and R4 are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.
  • The term “peptide” refers to molecules of 2 to 40 amino acids, with molecules of 3 to 20 amino acids preferred and those of 6 to 15 amino acids most preferred. Exemplary peptides may be randomly generated by any of the methods cited above, carried in a peptide library (e.g., a phage display library), or derived by digestion of proteins. [0067]
  • The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, [0068] E. coli display, ribosome display, RNA-peptide screening, chemical screening, and the like.
  • The term “pharmacologically active” means that a substance so described is determined to have activity that affects a medical parameter (e.g., blood pressure, blood cell count, cholesterol level) or disease state (e.g., cancer, autoimmune disorders). Thus, pharmacologically active peptides comprise agonistic or mimetic and antagonistic peptides as defined below. [0069]
  • The terms “-mimetic peptide” and “-agonist peptide” refer to a peptide having biological activity comparable to a protein (e.g., EPO, TPO, G-CSF) that interacts with a protein of interest. These terms further include peptides that indirectly mimic the activity of a protein of interest, such as by potentiating the effects of the natural ligand of the protein of interest; see, for example, the G-CSF-mimetic peptides listed in Tables 2 and 7. Thus, the term “EPO-mimetic peptide” comprises any peptides that can be identified or derived as described in Wrighton et al. (1996), [0070] Science 273: 458-63, Naranda et al. (1999), Proc. Natl. Acad. Sci. USA 96: 7569-74, or any other reference in Table 2 identified as having EPO-mimetic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.
  • The term “TPO-mimetic peptide” comprises peptides that can be identified or derived as described in Cwirla et al. (1997), [0071] Science 276: 1696-9, U.S. Pat. Nos. 5,869,451 and 5,932,946 and any other reference in Table 2 identifed as having TPO-mimetic subject matter, as well as the U.S. patent application, “Thrombopoietic Compounds,” filed on even date herewith and hereby incorporated by reference. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.
  • The term “G-CSF-mimetic peptide” comprises any peptides that can be identified or described in Paukovits et al. (1984), [0072] Hoppe-Seylers Z. Physiol. Chem. 365: 303-11 or any of the references in Table 2 identified as having G-CSF-mimetic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.
  • The term “CTLA4-mimetic peptide” comprises any peptides that can be identified or derived as described in Fukumoto et al. (1998), [0073] Nature Biotech. 16: 267-70. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.
  • The term “-antagonist peptide” or “inhibitor peptide” refers to a peptide that blocks or in some way interferes with the biological activity of the associated protein of interest, or has biological activity comparable to a known antagonist or inhibitor of the associated protein of interest. Thus, the term “TNF-antagonist peptide” comprises peptides that can be identified or derived as described in Takasaki et al. (1997), [0074] Nature Biotech. 15: 1266-70 or any of the references in Table 2 identified as having TNF-antagonistic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.
  • The terms “IL-1 antagonist” and “IL-1ra-mimetic peptide” comprises peptides that inhibit or down-regulate activation of the IL-1 receptor by IL-1. IL-1 receptor activation results from formation of a complex among IL-1, IL-1 receptor, and IL-1 receptor accessory protein. IL-1 antagonist or IL-1ra-mimetic peptides bind to IL-1, IL-1 receptor, or IL-1 receptor accessory protein and obstruct complex formation among any two or three components of the complex. Exemplary IL-1 antagonist or IL-1ra-mimetic peptides can be identified or derived as described in U.S. Pat. Nos. 5,608,035, 5,786,331, 5,880,096, or any of the references in Table 2 identified as having IL-1ra-mimetic or IL-1 antagonistic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries. [0075]
  • The term “VEGF-antagonist peptide” comprises peptides that can be identified or derived as described in Fairbrother (1998), [0076] Biochem. 37: 17754-64, and in any of the references in Table 2 identified as having VEGF-antagonistic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.
  • The term “MMP inhibitor peptide” comprises peptides that can be identified or derived as described in Koivunen (1999), [0077] Nature Biotech. 17: 768-74 and in any of the references in Table 2 identified as having MMP inhibitory subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.
  • Additionally, physiologically acceptable salts of the compounds of this invention are also encompassed herein. By “physiologically acceptable salts” is meant any salts that are known or later discovered to be pharmaceutically acceptable. Some specific examples are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate. [0078]
  • Structure of Compounds [0079]
  • In General. In the compositions of matter prepared in accordance with this invention, the peptide may be attached to the vehicle through the peptide's N-terminus or C-terminus. Thus, the vehicle-peptide molecules of this invention may be described by the following formula I: [0080]
  • (X1)a-F1-(X2)b  I
  • wherein: [0081]
  • F[0082] 1 is a vehicle (preferably an Fc domain);
  • X[0083] 1 and X2 are each independently selected from -(L1)c-P1, -(L1)c-P1-(L1)d-P2, -(L1)c-P1-(L2)d-P2-(L3)e-P3, and -(L1)c-P1-(L2)d-P2-(L3)e-P3-(L4)f-P4
  • P[0084] 1, P2, P3, and P4 are each independently sequences of pharmacologically active peptides;
  • L[0085] 1, L2, L3, and L4 are each independently linkers; and
  • a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1. [0086]
  • Thus, compound I comprises preferred compounds of the formulae [0087]
  • X1-F1  II
  • and multimers thereof wherein F[0088] 1 is an Fc domain and is attached at the C-terminus of X1;
  • F1-X2  III
  • and multimers thereof wherein F[0089] 1 is an Fc domain and is attached at the N-terminus of X2;
  • F1-(L1)c-P1  IV
  • and multimers thereof wherein F[0090] 1 is an Fc domain and is attached at the N-terminus of -(L1)c-P1; and
  • F-(L1)c-P1-(L2)d-P2  V
  • and multimers thereof wherein F[0091] 1 is an Fc domain and is attached at the N-terminus of -L1-P1-L2-P2.
  • Peptides. Any number of peptides may be used in conjunction with the present invention. Of particular interest are peptides that mimic the activity of EPO, TPO, growth hormone, G-CSF, GM-CSF, IL-1ra, leptin, CTLA4, TRAIL, TGF-α, and TGF-β. Peptide antagonists are also of interest, particularly those antagonistic to the activity of TNF, leptin, any of the interleukins (IL-1, 2, 3, . . . ), and proteins involved in complement activation (e.g., C3b). Targeting peptides are also of interest, including tumor-homing peptides, membrane-transporting peptides, and the like. All of these classes of peptides may be discovered by methods described in the references cited in this specification and other references. [0092]
  • Phage display, in particular, is useful in generating peptides for use in the present invention. It has been stated that affinity selection from libraries of random peptides can be used to identify peptide ligands for any site of any gene product. Dedman et al. (1993), [0093] J. Biol. Chem. 268: 23025-30. Phage display is particularly well suited for identifying peptides that bind to such proteins of interest as cell surface receptors or any proteins having linear epitopes. Wilson et al. (1998), Can. T. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins are extensively reviewed in Herz et al. (1997), T. Receptor & Signal Transduction Res. 17(5): 671-776, which is hereby incorporated by reference. Such proteins of interest are preferred for use in this invention.
  • A particularly preferred group of peptides are those that bind to cytokine receptors. Cytokines have recently been classified according to their receptor code. See Inglot (1997), [0094] Archivum Immunologiae et Therapiae Experimentalis 45: 353-7, which is hereby incorporated by reference. Among these receptors, most preferred are the CKRs (family I in Table 3). The receptor classification appears in Table 3.
    TABLE 3
    Cytokine Receptors Classified by Receptor Code
    Cytokines (ligands) Receptor Type
    family subfamily family subfamily
    I. Hematopoietic 1. IL-2, IL-4, IL-7, I. Cytokine R 1. shared γCr
    cytokines IL-9, IL-13, IL- (CKR)
    15
    2. IL-3, IL-5, GM- 2. shared GP
    CSF 140 βR
    3. IL-6, IL-11, IL- 3. 3.shared
    12, LIF, OSM, RP 130
    CNTF, leptin
    (OB)
    4. G-CSF, EPO, 4. “single
    TPO, PRL, GH chain” R
    5. IL-17, HVS-IL- 5. other Rc
    17
    II. IL-10 ligands IL-10, BCRF-1, II. IL-10 R
    HSV-IL-10
    III. Interferons 1. IFN-αl, α2, α4, III. Interferon R 1. IFNAR
    m, t, IFN-βd
    2. IFN-γ 2. IFNGR
    IV. IL-1 ligands 1. IL-1α, IL-1β, IL- IV. IL-1R
    1Ra
    V. TNF ligands 1. TNF-α, TNF-β V. NGF/TNF Re
    (LT), FAS1,
    CD40 L,
    CD30L, CD27 L
    VI. Chemokines 1. α chemokines: VI. Chemokine R 1. CXCR
    IL-8, GRO
    α, β, γ, IF-10,
    PF-4, SDF-1
    2. β chemokines: 2. CCR
    MIP1α, MIP1β,
    MCP-1, 2, 3, 4,
    RANTES,
    eotaxin
    3. γ chemokines: 3. CR
    lymphotactin 4. DARCf
    VII. Growth 1.1 SCF, M-CSF, VII. RKF 1. TK
    factors PDGF-AA, AB, sub-family
    BB, FLT-3L, 1.1 IgTK
    VEGF, SSV- III R
    PDGF
    1.2 FGFα, FGFβ 1.2 IgTK
    1.3 EGF, TGF-α, IV R
    VV-F19 (EGF- 1.3 Cysteine-
    like) rich TK-I
    1.4 IGF-I, IGF-II, 1.4 Cysteine
    Insulin rich TK-II
    1.5 NGF, BDNF, 1.5 Cysteine
    NT-3, NT-4g knot TK V
    2. TGF-β1, β2, β3 2. STK
    subfamilyh
  • Exemplary peptides for this invention appear in Tables 4 through 20 below. These peptides may be prepared by methods disclosed in the art. Single letter amino acid abbreviations are used. The X in these sequences (and throughout this specification, unless specified otherwise in a particular instance) means that any of the 20 naturally occurring amino acid residues may be present. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers, and a few tandem-linked examples are provided in the table. Linkers are listed as “A” and may be any of the linkers described herein. Tandem repeats and linkers are shown separated by dashes for clarity. Any peptide containing a cysteinyl residue may be cross-linked with another Cys-containing peptide, either or both of which may be linked to a vehicle. A few cross-linked examples are provided in the table. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well; see, for example, EPO-mimetic peptides in Table 5. A few examples of intrapeptide disulfide-bonded peptides are specified in the table. Any of these peptides may be derivatized as described herein, and a few derivatized examples are provided in the table. Derivatized peptides in the tables are exemplary rather than limiting, as the associated underivatized peptides may be employed in this invention, as well. For derivatives in which the carboxyl terminus may be capped with an amino group, the capping amino group is shown as —NH[0095] 2. For derivatives in which amino acid residues are substituted by moieties other than amino acid residues, the substitutions are denoted by σ, which signifies any of the moieties described in Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9 and Cuthbertson et al. (1997), T. Med. Chem. 40: 2876-82, which are incorporated by reference. The J substituent and the Z substituents (Z5, Z6, . . . Z40) are as defined in U.S. Pat. Nos. 5,608,035, 5,786,331, and 5,880,096, which are incorporated by reference. For the EPO-mimetic sequences (Table 5), the substituents X2 through X11 and the integer “n” are as defined in WO 96/40772, which is incorporated by reference. The substituents “Ψ,” “Θ,” and “+” are as defined in Sparks et al. (1996), Proc. Natl. Acad. Sci. 93: 1540-4, which is hereby incorporated by reference. X4, X5, X6, and X7 are as defined in U.S. Pat. No. 5,773,569, which is hereby incorporated by reference, except that: for integrin-binding peptides, X1, X2, X3, X4, X5, X6, X7, and X8 are as defined in International applications WO 95/14714, published Jun. 1, 1995 and WO 97/08203, published Mar. 6, 1997, which are also incorporated by reference; and for VIP-mimetic peptides, X1, X1′, X1″, X2, X3, X4, X5, X6 and Z and the integers m and n are as defined in WO 97/40070, published Oct. 30, 1997, which is also incorporated by reference. Xaa and Yaa below are as defined in WO 98/09985, published Mar. 12, 1998, which is incorporated by reference. AA1, AA2, AB1, AB2, and AC are as defined in International application WO 98/53842, published Dec. 3, 1998, which is incorporated by reference. X1, X2, X3, and X4 in Table 17 only are as defined in European application EP 0 911 393, published Apr. 28, 1999. Residues appearing in boldface are D-amino acids. All peptides are linked through peptide bonds unless otherwise noted. Abbreviations are listed at the end of this specification. In the “SEQ ID NO.” column, “NR” means that no sequence listing is required for the given sequence.
    TABLE 4
    IL-1 antagonist peptide sequences
    Sequence/structure SEQ ID NO:
    Z11Z7Z8QZ5Y4Z9Z10 212
    XXQZ5YZ6XX 907
    Z7XQZ5YZ6XX 908
    Z7Z8QZ5YZ6Z9Z10 909
    Z11Z7Z8QZ5YZ6Z9Z10 910
    Z12Z13Z14Z15Z16Z17Z18Z19Z20Z21Z22Z11Z7Z8QZ5YZ6Z9Z10L 917
    Z23NZ24Z39Z25Z26Z27Z28Z29Z30Z40 979
    TANVSSFEWTPYYWQPYALPL 213
    SWTDYGYWQPYALPISGL 214
    ETPFTWEESNAYYWQPYALPL 215
    ENTYSPNWADSMYWQPYALPL 216
    SVGEDHNFWTSEYWQPYALPL 217
    DGYDRWRQSGERYWQPYALPL 218
    FEWTPGYWQPY 219
    FEWTPGYWQHY 220
    FEWTPGWYQJY 221
    AcFEWTPGWYQJY 222
    FEWTPGWpYQJY 223
    FAWTPGYWQJY 224
    FEWAPGYWQJY 225
    FEWVPGYWQJY 226
    FEWTPGYWQJY 227
    AcFEWTPGYWQJY 228
    FEWTPaWYQJY 229
    FEWTPSarWYQJY 230
    FEWTPGYYQPY 231
    FEWTPGWWQPY 232
    FEWTPNYWQPY 233
    FEWTPvYWQJY 234
    FEWTPecGYWQJY 235
    FEWIPAibYWQJY 236
    FEWTSarGYWQJY 237
    FEWTPGYWQPY 238
    FEWTPGYWQHY 239
    FEWTPGWYQJY 240
    AcFEWTPGWYQJY 241
    FEWTPGW-pY-QJY 242
    FAWTPGYWQJY 243
    FEWAPGYWQJY 244
    FEWVPGYWQJY 245
    FEWTPGYWQJY 246
    AcFEWTPGYWQJY 247
    FEWTPAWYQJY 248
    FEWTPSarWYQJY 249
    FEWTPGYYQPY 250
    FEWTPGWWQPY 251
    FEWTPNYWQPY 252
    FEWTPVYWQJY 253
    FEWTPecGYWQJY 254
    FEWTPAibYWQJY 255
    FEWTSarGYWQJY 256
    FEWTPGYWQPYALPL 257
    1NapEWTPGYYQJY 258
    YEWTPGYYQJY 259
    FEWVPGYYQJY 260
    FEWTPSYYQJY 261
    FEWTPNYYQJY 262
    TKPR 263
    RKSSK 264
    RKQDK 265
    NRKQDK 266
    RKQDKR 267
    ENRKQDKRF 268
    VTKFYF 269
    VTKFY 270
    VTDFY 271
    SHLYWQPYSVQ 671
    TLVYWQPYSLQT 672
    RGDYWQPYSVQS 673
    VHVYWQPYSVQT 674
    RLVYWQPYSVQT 675
    SRVWFQPYSLQS 676
    NMVYWQPYSIQT 677
    SVVFWQPYSVQT 678
    TFVYWQPYALPL 679
    TLVYWQPYSIQR 680
    RLVYWQPYSVQR 681
    SPVFWQPYSIQI 682
    WIEWWQPYSVQS 683
    SLIYWQPYSLQM 684
    TRLYWQPYSVQR 685
    RCDYWQPYSVQT 686
    MRVFWQPYSVQN 687
    KIVYWQPYSVQT 688
    RHLYWQPYSVQR 689
    ALVWWQPYSEQI 690
    SRVWFQPYSLQS 691
    WEQPYALPLE 692
    QLVWWQPYSVQR 693
    DLRYWQPYSVQV 694
    ELVWWQPYSLQL 695
    DLVWWQPYSVQW 696
    NGNYWQPYSFQV 697
    ELVYWQPYSIQR 698
    ELMYWQPYSVQE 699
    NLLYWQPYSMQD 700
    GYEWYQPYSVQR 701
    SRVWYQPYSVQR 702
    LSEQYQPYSVQR 703
    GGGWWQPYSVQR 704
    VGRWYQPYSVQR 705
    VHVYWQPYSVQR 706
    QARWYQPYSVQR 707
    VHVYWQPYSVQT 708
    RSVYWQPYSVQR 709
    TRVWFQPYSVQR 710
    GRIWFQPYSVQR 711
    GRVWFQPYSVQR 712
    ARTWYQPYSVQR 713
    ARVWWQPYSVQM 714
    RLMFYQPYSVQR 715
    ESMWYQPYSVQR 716
    HFGWWQPYSVHM 717
    ARFWWQPYSVQR 718
    RLVYWQ PYAPIY 719
    RLVYWQ PYSYQT 720
    RLVYWQ PYSLPI 721
    RLVYWQ PYSVQA 722
    SRVWYQ PYAKGL 723
    SRVWYQ PYAQGL 724
    SRVWYQ PYAMPL 725
    SRVWYQ PYSVQA 726
    SRVWYQ PYSLGL 727
    SRVWYQ PYAREL 728
    SRVWYQ PYSRQP 729
    SRVWYQ PYFVQP 730
    EYEWYQ PYALPL 731
    IPEYWQ PYALPL 732
    SRIWWQ PYALPL 733
    DPLFWQ PYALPL 734
    SRQWVQ PYALPL 735
    IRSWWQ PYALPL 736
    RGYWQ PYALPL 737
    RLLWVQ PYALPL 738
    EYRWFQ PYALPL 739
    DAYWVQ PYALPL 740
    WSGYFQ PYALPL 741
    NIEFWQ PYALPL 742
    TRDWVQ PYALPL 743
    DSSWYQ PYALPL 744
    IGNWYQ PYALPL 745
    NLRWDQ PYALPL 746
    LPEFWQ PYALPL 747
    DSYWWQ PYALPL 748
    RSQYYQ PYALPL 749
    ARFWLQ PYALPL 750
    NSYFWQ PYALPL 751
    RFMYWQPYSVQR 752
    AHLFWQPYSVQR 753
    WWQPYALPL 754
    YYQPYALPL 755
    YFQPYALGL 756
    YWYQPYALPL 757
    RWWQPYATPL 758
    GWYQPYALGF 759
    YWYQPYALGL 760
    IWYQPYAMPL 761
    SNMQPYQRLS 762
    TFVYWQPY AVGLPAAETACN 763
    TFVYWQPY SVQMTITGKVTM 764
    TFVYWQPY SSHXXVPXGFPL 765
    TFVYWQPY YGNPQWAIHVRH 766
    TFVYWQPY VLLELPEGAVRA 767
    TFVYWQPY VDYVWPIPIAQV 768
    GWYQPYVDGWR 769
    RWEQPYVKDGWS 770
    EWYQPYALGWAR 771
    GWWQPYARGL 772
    LFEQPYAKALGL 773
    GWEQPYARGLAG 774
    AWVQPYATPLDE 775
    MWYQPYSSQPAE 776
    GWTQPYSQQGEV 777
    DWFQPYSIQSDE 778
    PWIQPYARGFG 779
    RPLYWQPYSVQV 780
    TLIYWQPYSVQI 781
    RFDYWQPYSDQT 782
    WHQFVQPYALPL 783
    EWDS VYWQPYSVQ TLLR 784
    WEQN VYWQPYSVQ SFAD 785
    SDV VYWQPYSVQ SLEM 786
    YYDG VYWQPYSVQ VMPA 787
    SDIWYQ PYALPL 788
    QRIWWQ PYALPL 789
    SRIWWQ PYALPL 790
    RSLYWQ PYALPL 791
    TIIWEQ PYALPL 792
    WETWYQ PYALPL 793
    SYDWEQ PYALPL 794
    SRIWCQ PYALPL 795
    EIMFWQ PYALPL 796
    DYVWQQ PYALPL 797
    MDLLVQ WYQPYALPL 798
    GSKVIL WYQPYALPL 799
    RQGANI WYQPYALPL 800
    GGGDEP WYQPYALPL 801
    SQLERT WYQPYALPL 802
    ETWVRE WYQPYALPL 803
    KKGSTQ WYQPYALPL 804
    LQARMN WYQPYALPL 805
    EPRSQK WYQPYALPL 806
    VKQKWR WYQPYALPL 807
    LRRHDV WYQPYALPL 808
    RSTASI WYQPYALPL 809
    ESKEDQ WYQPYALPL 810
    EGLTMK WYQPYALPL 811
    EGSREG WYQPYALPL 812
    VIEWWQ PYALPL 813
    VWYWEQ PYALPL 814
    ASEWWQ PYALPL 815
    FYEWWQ PYALPL 816
    EGWWVQ PYALPL 817
    WGEWLQ PYALPL 818
    DYVWEQ PYALPL 819
    AHTWWQ PYALPL 820
    FIEWFQ PYALPL 821
    WLAWEQ PYALPL 822
    VMEWWQ PYALPL 823
    ERMWQ PYALPL 824
    NXXWXX PYALPL 825
    WGNWYQ PYALPL 826
    TLYWEQ PYALPL 827
    VWRWEQ PYALPL 828
    LLWTQ PYALPL 829
    SRIWXX PYALPL 830
    SDIWYQ PYALPL 831
    WGYYXX PYALPL 832
    TSGWYQ PYALPL 833
    VHPYXX PYALPL 834
    EHSYFQ PYALPL 835
    XXIWYQ PYALPL 836
    AQLHSQ PYALPL 837
    WANWFQ PYALPL 838
    SRLYSQ PYALPL 839
    GVTFSQ PYALPL 840
    SIVWSQ PYALPL 841
    SRDLVQ PYALPL 842
    HWGH VYWQPYSVQ DDLG 843
    SWHS VYWQPYSVQ SVPE 844
    WRDS VYWQPYSVQ PESA 845
    TWDA VYWQPYSVQ KWLD 846
    TPPW VYWQPYSVQ SLDP 847
    YWSS VYWQPYSVQ SVHS 848
    YWY QPY ALGL 849
    YWY QPY ALPL 850
    EWI QPY ATGL 851
    NWE QPY AKPL 852
    AFY QPY ALPL 853
    FLY QPY ALPL 854
    VCK QPY LEWC 855
    ETPFTWEESNAYYWQPYALPL 856
    QGWLTWQDSVDMYWQPYALPL 857
    FSEAGYTWPENTYWQPYALPL 858
    TESPGGLDWAKIYWQPYALPL 859
    DGYDRWRQSGERYWQPYALPL 860
    TANVSSFEWTPGYWQPYALPL 861
    SVGEDHNFWTSE YWQPYALPL 862
    MNDQTSEVSTFP YWQPYALPL 863
    SWSEAFEQPRNL YWQPYALPL 864
    QYAEPSALNDWG YWQPYALPL 865
    NGDWATADWSNY YWQPYALPL 866
    THDEHI YWQPYALPL 867
    MLEKTYTTWTPG YWQPYALPL 868
    WSDPLTRDADL YWQPYALPL 869
    SDAFTTQDSQAM YWQPYALPL 870
    GDDAAWRTDSLT YWQPYALPL 871
    AIIRQLYRWSEM YWQPYALPL 872
    ENTYSPNWADSM YWQPYALPL 873
    MNDQTSEVSTFP YWQPYALPL 874
    SVGEDHNFWTSE YWQPYALPL 875
    QTPFTWEESNAY YWQPYALPL 876
    ENPFTWQESNAY YWQPYALPL 877
    VTPFTWEDSNVF YWQPYALPL 878
    QIPFTWEQSNAY YWQPYALPL 879
    QAPLTWQESAAY YWQPYALPL 880
    EPTFTWEESKAT YWQPYALPL 881
    TTTLTWEESNAY YWQPYALPL 882
    ESPLTWEESSAL YWQPYALPL 883
    ETPLTWEESNAY YWQPYALPL 884
    EATFTWAESNAY YWQPYALPL 885
    EALFTWKESTAY YWQPYALPL 886
    STP-TWEESNAY YWQPYALPL 887
    ETPFTWEESNAY YWQPYALPL 888
    KAPFTWEESQAY YWQPYALPL 889
    STSFTWEESNAY YWQPYALPL 890
    DSTFTWEESNAY YWQPYALPL 891
    YIPFTWEESNAY YWQPYALPL 892
    QTAFTWEESNAY YWQPYALPL 893
    ETLFTWEESNAT YWQPYALPL 894
    VSSFTWEESNAY YWQPYALPL 895
    QPYALPL 896
    Py-1-NapPYQJYALPL 897
    TANVSSFEWTPG YWQPYALPL 898
    FEWTPGYWQPYALPL 899
    FEWTPGYWQJYALPL 900
    FEWTPGYYQJYALPL 901
    ETPFTWEESNAYYWQPYALPL 902
    FTWEESNAYYWQJYALPL 903
    ADVL YWQPYA PVTLWV 904
    GDVAE YWQPYA LPLTSL 905
    SWTDYG YWQPYA LPISGL 906
    FEWTPGYWQPYALPL 911
    FEWTPGYWQJYALPL 912
    FEWTPGWYQPYALPL 913
    FEWTPGWYQJYALPL 914
    FEWTPGYYQPYALPL 915
    FEWTPGYYQJYALPL 916
    TANVSSFEWTPGYWQPYALPL 918
    SWTDYGYWQPYALPISGL 919
    ETPFTWEESNAYYWQPYALPL 920
    ENTYSPNWADSMYWQPYALPL 921
    SVGEDHNFWTSEYWQPYALPL 922
    DGYDRWRQSGERYWQPYALPL 923
    FEWTPGYWQPYALPL 924
    FEWTPGYWQPY 925
    FEWTPGYWQJY 926
    EWTPGYWQPY 927
    FEWTPGWYQJY 928
    AEWTPGYWQJY 929
    FAWTPGYWQJY 930
    FEATPGYWQJY 931
    FEWAPGYWQJY 932
    FEWTAGYWQJY 933
    FEWTPAYWQJY 934
    FEWTPGAWQJY 935
    FEWTPGYAQJY 936
    FEWTPGYWQJA 937
    FEWTGGYWQJY 938
    FEWTPGYWQJY 939
    FEWTJGYWQJY 940
    FEWTPecGYWQJY 941
    FEWTPAibYWQJY 942
    FEWTPSarWYQJY 943
    FEWTSarGYWQJY 944
    FEWTPNYWQJY 945
    FEWTPVYWQJY 946
    FEWTVPYWQJY 947
    AcFEWTPGWYQJY 948
    AcFEWTPGYWQJY 949
    INap-EWTPGYYQJY 950
    YEWTPGYYQJY 951
    FEWVPGYYQJY 952
    FEWTPGYYQJY 953
    FEWTPsYYQJY 954
    FEWTPnYYQJY 955
    SHLY-Nap-QPYSVQM 956
    TLVY-Nap-QPYSLQT 957
    RGDY-Nap-QPYSVQS 958
    NMVY-Nap-QPYSIQT 959
    VYWQPYSVQ 960
    VY-Nap-QPYSVQ 961
    TFVYWQJYALPL 962
    FEWTPGYYQJ-Bpa 963
    XaaFEWTPGYYQJ-Bpa 964
    FEWTPGY-Bpa-QJY 965
    AcFEWTPGY-Bpa-QJY 966
    FEWTPG-Bpa-YQJY 967
    AcFEWTPG-Bpa-YQJY 968
    AcFE-Bpa-TPGYYQJY 969
    AcFE-Bpa-TPGYYQJY 970
    Bpa-EWTPGYYQJY 971
    AcBpa-EWTPGYYQJY 972
    VYWQPYSVQ 973
    RLVYWQPYSVQR 974
    RLVY-Nap-QPYSVQR 975
    RLDYWQPYSVQR 976
    RLVWFQPYSVQR 977
    RLVYWQPYSIQR 978
    DNSSWYDSFLL 980
    DNTAWYESFLA 981
    DNTAWYENFLL 982
    PARE DNTAWYDSFLI WC 983
    TSEY DNTTWYEKFLA SQ 984
    SQIP DNTAWYQSFLL HG 985
    SPFI DNTAWYENFLL TY 986
    EQIY DNTAWYDHFLL SY 987
    TPFI DNTAWYENFLL TY 988
    TYTY DNTAWYERFLM SY 989
    TMTQ DNTAWYENFLL SY 990
    TI DNTAWYANLVQ TYPQ 991
    TI DNTAWYERFLA QYPD 992
    HI DNTAWYENFLL TYTP 993
    SQ DNTAWYENFLL SYKA 994
    QI DNTAWYERFLL QYNA 995
    NQ DNTAWYESFLL QYNT 996
    TI DNTAWYENFLL NHNL 997
    HY DNTAWYERFLQ QGWH 998
    ETPFTWEESNAYYWQPYALPL 999
    YIPFTWEESNAYYWQPYALPL 1000
    DGYDRWRQSGERYWQPYALPL 1001
    pY-INap-pY-QJYALPL 1002
    TANVSSFEWTPGYWQPYALPL 1003
    FEWTPGYWQJYALPL 1004
    FEWTPGYWQPYALPLSD 1005
    FEWTPGYYQJYALPL 1006
    FEWTPGYWQJY 1007
    AcFEWTPGYWQJY 1008
    AcFEWTPGWYQJY 1009
    AcFEWTPGYYQJY 1010
    AcFEWTPaYWQJY 1011
    AcFEWTPaWYQJY 1012
    AcFEWTPaYYQJY 1013
    FEWTPGYYQJYALPL 1014
    FEWTPGYWQJYALPL 1015
    FEWTPGWYQJYALPL 1016
    TANVSSFEWTPGYWQPYALPL 1017
    AcFEWTPGYWQJY 1018
    AcFEWTPGWYQJY 1019
    AcFEWTPGYYQJY 1020
    AcFEWTPAYWQJY 1021
    AcFEWTPAWYQJY 1022
    AcFEWTPAYYQJY 1023
  • [0096]
    TABLE 5
    EPO-mimetic peptide sequences
    SEQ
    Sequence/structure ID NO:
    YXCXXGPXTWXCXP  83
    YXCXXGPXTWXCXP-YXCXXGPXTWXCXP  84
    YXCXXGPXTWXCXP-Λ-YXCXXGPXTWXCXP  85
    Figure US20040071712A1-20040415-C00001
     86     86
    GGTYSCHFGPLTWVCKPQGG  87
    GGDYHCRMGPLTWVCKPLGG  88
    GGVYACRMGPITWVCSPLGG  89
    VGNYMCHFGPITWVCRPGGG  90
    GGLYLCRFGPVTWDCGYKGG  91
    GGTYSCHFGPLTWVCKPQGG-  92
    GGTYSCHFGPLTWVCKPQGG
    GGTYSCHFGPLTWVCKPQGG-Λ-  93
    GGTYSCHFGPLTWVCKPQGG
    GGTYSCHFGPLTWVCKPQGGSSK  94
    GGTYSCHFGPLTWVCKPQGGSSK-  95
    GGTYSCHFGPLTWVCKPQGGSSK
    GGTYSCHFGPLTWVCKPQGGSSK-Λ-  96
    GGTYSCHFGPLTWVCKPQGGSSK
    Figure US20040071712A1-20040415-C00002
     97     97
    GGTYSCHFGPLTWVCKPQGGSSK(-Λ-biotin)  98
    CX4X5GPX6TWX7C 421
    GGTYSCHGPLTWVCKPQGG 422
    VGNYMAHMGPITWVGRPGG 423
    GGPHHVYACRMGPLTWIC 424
    GGTYSCHFGPLTWVCKPQ 425
    GGLYACHMGPMTWVCQPLRG 426
    TIAQYICYMGPETWECRPSPKA 427
    YSCHFGPLTWVCK 428
    YCHFGPLTWVC 429
    X3X4X5GPX6TWX7X8 124
    YX2X3X4X5GPX6TWX7X8 461
    X1YX2X3X4X5GPX6TWX7X8X9X10X11 419
    X1YX2CX4X5GPX6TWX7CX9X10X11 420
    GGLYLCRFGPVTWDCGYKGG 1024 
    GGTYSCHFGPLTWVCKPQGG 1025 
    GGDYHCRMGPLTWVCKPLGG 1026 
    VGNYMCHFGPITWVCRPGGG 1029 
    GGVYACRMGPITWVCSPLGG 1030 
    VGNYMAHMGPITWVCRPGG 1035 
    GGTYSCHFGPLTWVCKPQ 1036 
    GGLYACHMGPMTWVCQPLRG 1037 
    TIAQYICYMGPETWECRPSPKA 1038 
    YSCHFGPLTWVCK 1039 
    YCHFGPLTWVC 1040 
    SCHFGPLTWVCK 1041 
    (AX2)nX3X4X5GPX6TWX7X8 1042 
  • [0097]
    TABLE 6
    TPO-mimetic peptide sequences
    SEQ
    Sequence/structure ID NO:
    IEGPTLRQWLAARA 13
    IEGPTLRQWLAAKA 24
    IEGPTLREWLAARA 25
    IEGPTLRQWLAARA-Λ-IEGPTLRQWLAARA 26
    IEGPTLRQWLAAKA-Λ-IEGPTLRQWLAAKA 27
    Figure US20040071712A1-20040415-C00003
    28
    IEGPTLRQWLAARA-Λ-K(BrAc)-Λ-IEGPTLRQWLAARA 29
    IEGPTLRQWLAARA-Λ-K(PEG)-Λ-IEGPTLRQWLAARA 30
    Figure US20040071712A1-20040415-C00004
    31  31
    Figure US20040071712A1-20040415-C00005
    32  32
    VRDQIXXXL 33
    TLREWL 34
    GRVRDQVAGW 35
    GRVKDQIAQL 36
    GVRDQVSWAL 37
    ESVREQVMKY 38
    SVRSQISASL 39
    GVRETVYRHM 40
    GVREVIVMHML 41
    GRVRDQIWAAL 42
    AGVRDQILIWL 43
    GRVRDQIMLSL 44
    GRVRDQI(X)3L 45
    CTLRQWLQGC 46
    CTLQEFLEGC 47
    CTRTEWLHGC 48
    CTLREWLHGGFC 49
    CTLREWVFAGLC 50
    CTLRQWLILLGMC 51
    CTLAEFLASGVEQC 52
    CSLQEFLSHGGYVC 53
    CTLREFLDPTTAVC 54
    CTLKEWLVSHEVWC 55
    CTLREWL(X)2-6C 56-60
    REGPTLRQWM 61
    EGPTLRQWLA 62
    ERGPFWAKAC 63
    REGPRCVMWM 64
    CGTEGPTLSTWLDC 65
    CEQDGPTLLEWLKC 66
    CELVGPSLMSWLTC 67
    CLTGPFVTQWLYEC 68
    CRAGPTLLEWLTLC 69
    CADGPTLREWISFC 70
    C(X)1-2EGPTLREWL(X)1-2C 71-74
    GGCTLREWLHGGFCGG 75
    GGCADGPTLREWISFCGG 76
    GNADGPTLRQWLEGRRPKN 77
    LAIEGPTLRQWLHGNGRDT 78
    HGRVGPTLREWKTQVATKK 79
    TIKGPTLRQWLKSREHTS so
    ISDGPTLKEWLSVTRGAS 81
    SIEGPTLREWLTSRTPHS 82
  • [0098]
    TABLE 7
    G-CSF-mimetic peptide sequences
    SEQ
    Sequence/structure ID NO:
    EEDCK  99
    Figure US20040071712A1-20040415-C00006
     99   99
    EEDσK 100
    Figure US20040071712A1-20040415-C00007
    100  100
    pGluEDσK 101
    Figure US20040071712A1-20040415-C00008
    101  101
    PicSDσK 102
    Figure US20040071712A1-20040415-C00009
    102  102
    EEDCK-Λ-EEDCK 103
    EEDXK-Λ-EEDXK 104
  • [0099]
    TABLE 8
    TNF-antagonist peptide sequences
    SEQ
    Sequence/structure ID NO:
    YCFTASENHCY 106
    YCFTNSENHCY 107
    YCFTRSENHGY 108
    FCASENHCY 109
    YCASENHCY 110
    FCNSENHCY 111
    FCNSENRCY 112
    FCNSVENRCY 113
    YCSQSVSNDCF 114
    FCVSNDRCY 115
    YCRKELGQVCY 116
    YCKEPGQCY 117
    YCRKEMGCY 118
    FCRKEMGCY 119
    YCWSQNLCY 120
    YCELSQYLCY 121
    YCWSQNYCY 122
    YCWSQYLCY 123
    DFLPHYKNTSLGHRP 1085 
    Figure US20040071712A1-20040415-C00010
    NR
  • [0100]
    TABLE 9
    Integrin-binding peptide sequences
    Sequence/structure SEQ ID NO:
    RX1ETX2WX3 441
    RX1ETX2WX3 442
    RGDGX 443
    CRGDGXC 444
    CX1X2RLDX3X4C 445
    CARRLDAPC 446
    CPSRLDSPC 447
    X1X2X3RGDX4X5X6 448
    CX2CRGDCX5C 449
    CDCRGDCFC 450
    CDGRGDCLC 451
    CLCRGDCIC 452
    X1X2DDX4X5X7X8 453
    X1X2X3DDX4X5X6X7X8 454
    CWDDGWLC 455
    CWDDLWWLC 456
    CWDDGLMC 457
    CWDDGWMC 458
    CSWDDGWLC 459
    CPDDLWWLC 460
    NGR NR
    GSL NR
    RGD NR
    CGRECPRLCQSSC 1071
    CNGRCVSGCAGRC 1072
    CLSGSLSC 1073
    RGD NR
    NGR NR
    GSL NR
    NGRAHA 1074
    CNGRC 1075
    CDCRGDCFC 1076
    CGSLVRC 1077
    DLXXL 1043
    RTDLDSLRTYTL 1044
    RTDLDSLRTY 1053
    RTDLDSLRT 1054
    RTDLDSLR 1078
    GDLDLLKLRLTL 1079
    GDLHSLRQLLSR 1080
    RDDLHMLRLQLW 1081
    SSDLHALKKRYG 1082
    RGDLKQLSELTW 1083
    RGDLAALSAPPV 1084
  • [0101]
    TABLE 10
    Selectin antagonist peptide sequences
    Sequence/structure SEQ ID NO:
    DITWDQLWDLMK 147
    DITWDELWKIMN 148
    DYTWFELWDMMQ 149
    QITWAQLWNMMK 150
    DMTWHDLWTLMS 151
    DYSWHDLWEMMS 152
    EITWDQLWEVMN 153
    HVSWEQLWDIMN 154
    HITWDQLWRIMT 155
    RNMSWLELWEHMK 156
    AEWTWDQLWHVMNPAESQ 157
    HRAEWLALWEQMSP 158
    KKEDWLALWRIMSV 159
    ITWDQLWDLMK 160
    DITWDQLWDLMK 161
    DITWDQLWDLMK 162
    DITWDQLWDLMK 163
    CQNRYTDLVAIQNKNE 462
    AENWADNEPNNKRNNED 463
    RKNNKTWTWVGTKKALTNE 464
    KKALTNEAENWAD 465
    CQXRYTDLVAIQNKXE 466
    RKXNXXWTWVGTXKXLTEE 467
    AENWADGEPNNKXNXED 468
    CXXXYTXLVAIQNKXE 469
    RKXXXXWXWVGTXKXLTXE 470
    AXNWXXXEPNNXXXED 471
    XKXKTXEAXNWXX 472
  • [0102]
    TABLE 11
    Antipathogenic peptide sequences
    Sequence/structure SEQ ID NO:
    GFFALIPKIISSPLFKTLLSAVGSALSSSGGQQ 503
    GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE 504
    GFFALIPKIISSPLFKTLLSAV 505
    GFFALIPKIISSPLFKTLLSAV 506
    KGFFALIPKIISSPLFKTLLSAV 507
    KKGFFALIPKIISSPLFKTLLSAV 508
    KKGFFALIPKIISSPLFKTLLSAV 509
    GFFALIPKIIS 510
    GIGAVLKVLTTGLPALISWIKRKRQQ 511
    GIGAVLKVLTTGLPALISWIKRKRQQ 512
    GIGAVLKVLTTGLPALISWIKRKRQQ 513
    GIGAVLKVLTTGLPALISWIKR 514
    AVLKVLTTGLPALISWIKR 515
    KLLLLLKLLLLK 516
    KLLLKLLLKLLK 517
    KLLLKLKLKLLK 518
    KKLLKLKLKLKK 519
    KLLLKLLLKLLK 520
    KLLLKLKLKLLK 521
    KLLLLK 522
    KLLLKLLK 523
    KLLLKLKLKLLK 524
    KLLLKLKLKLLK 525
    KLLLKLKLKLLK 526
    KAAAKAAAKAAK 527
    KVVVKVVVKVVK 528
    KVVVKVKVKVVK 529
    KVVVKVKVKVK 530
    KVVVKVKVKVVK 531
    KLILKL 532
    KVLHLL 533
    LKLRLL 534
    KPLHLL 535
    KLILKLVR 536
    KVFHLLHL 537
    HKFRILKL 538
    KPFHILHL 539
    KIIIKIKIKIIK 540
    KIIIKIKIKIIK 541
    KIIIKIKIKIIK 542
    KIPIKIKIKIPK 543
    KIPIKIKIKIVK 544
    RIIIRIRIRIIR 545
    RIIIRIRIRIIR 546
    RIIIRIRIRIIR 547
    RIVIRIRIRLIR 548
    RIIVRIRLRIIR 549
    RIGIRLRVRIIR 550
    KIVIRIRIRLIR 551
    RIAVKWRLRFIK 552
    KIGWKLRVRIIR 553
    KKIGWLIIRVRR 554
    RIVIRIRIRLIRIR 555
    RIIVRIRLRIIRVR 556
    RIGIRLRVRIIRRV 557
    KIVIRIRARLIRIRIR 558
    RIIVKIRLRIIKKIRL 559
    KIGIKARVRIIRVKII 560
    RIIVHIRLRIIHHIRL 561
    HIGIKAHVRIIRVHII 562
    RIYVKIHLRYIKKIRL 563
    KIGHKARVHIIRYKII 564
    RIYVKPHPRYIKKIRL 565
    KPGHKARPHIIRYKII 566
    KIVIRIRIRLIRIRIRKIV 567
    RIIVKIRLRIIKKIRLIKK 568
    KIGWKLRVRIIRVKIGRLR 569
    KIVIRIRIRLIRIRIRKIVKVKRIR 570
    RFAVKIRLRIIKKIRLIKKIRKRVIK 571
    KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK 572
    RIYVKPHPRYIKKIRL 573
    KPGHKARPHIIRYKII 574
    KIVIRIRIRLIRIRIRKIV 575
    RIIVKIRLRIIKKIRLIKK 576
    RIYVSKISIYIKKIRL 577
    KIVIFTRIRLTSIRIRSIV 578
    KPIHKARPTIIRYKMI 579
    cyclicCKGFFALIPKIISSPLFKTLLSAVC 580
    CKKGFFALIPKIISSPLFKTLLSAVC 581
    CKKKGFFALIPKIISSPLFKTLLSAVC 582
    CyclicCRIVIRIRIRLIRIRC 583
    CyclicCKPGHKARPHIIRYKIIC 584
    CyclicCRFAVKIRLRIIKKIRLIKKIRKAVIKC 585
    KLLLKLLL KLLKC 586
    KLLLKLLLKLLK 587
    KLLLKLKLKLLKC 588
    KLLLKLLLKLLK 589
  • [0103]
    TABLE 12
    VIP-mimetic peptide sequences
    SEQ
    Sequence/structure ID NO:
    HSDAVFYDNYTR LRKQMAVKKYLN SILN 590
    Nle HSDAVFYDNYTR LRKQMAVKKYLN SILN 591
    X1X1′X1″X2 592
    X3 S X4 LN 593
    Figure US20040071712A1-20040415-C00011
    594
    KKYL 595
    NSILN 596
    KKYL 597
    KKYA 598
    AVKKYL 599
    NSILN 600
    KKYV 601
    SILauN 602
    KKYLNle 603
    NSYLN 604
    NSIYN 605
    KKYLPPNSILN 606
    LauKKYL 607
    CapKKYL 608
    KYL NR
    KKYNle 609
    VKKYL 610
    LNSILN 611
    YLNSILN 612
    KKYLN 613
    KKYLNS 614
    KKYLNSI 615
    KKYLNSIL 616
    KKYL 617
    KKYDA 618
    AVKKYL 619
    NSILN 620
    KKYV 621
    SILauN 622
    NSYLN 623
    NSIYN 624
    KKYLNle 625
    KKYLPPNSILN 626
    KKYL 627
    KKYDA 628
    AVKKYL 629
    NSILN 630
    KKYV 631
    SILauN 632
    LauKKYL 633
    CapKKYL 634
    KYL NR
    KYL NR
    KKYNle 635
    VKKYL 636
    LNSILN 637
    YLNSILN 638
    KKYLNle 639
    KKYLN 640
    KKYLNS 641
    KKYLNSI 642
    KKYLNSIL 643
    KKKYLD 644
    cyclicCKKYLC 645
    Figure US20040071712A1-20040415-C00012
    646
    KKYA 647
    WWTDTGLW 648
    WWTDDGLW 649
    WWDTRGLWVWTI 650
    FWGNDGIWLESG 651
    DWDQFGLWRGAA 652
    RWDDNGLWVVVL 653
    SGMWSHYGIWMG 654
    GGRWDQAGLWVA 655
    KLWSEQGIWMGE 656
    CWSMHGLWLC 657
    GCWDNTGIWVPC 658
    DWDTRGLWVY 659
    SLWDENGAWI 660
    KWDDRGLWMH 661
    QAWNERGLWT 662
    QWDTRGLWVA 663
    WNVHGIWQE 664
    SWDTRGLWVE 665
    DWDTRGLWVA 666
    SWGRDGLWIE 667
    EWTDNGLWAL 668
    SWDEKGLWSA 669
    SWDSSGLWMD 670
  • [0104]
    TABLE 13
    Mdm/hdm antagonist peptide sequences
    Sequence/structure SEQ ID NO:
    TFSDLW 130
    QETFSDLWKLLP 131
    QPTFSDLWKLLP 132
    QETFSDYWKLLP 133
    QPTFSDYWKLLP 134
    MPRFMDYWEGLN 135
    VQNFIDYWTQQF 136
    TGPAFTHYWATF 137
    IDRAPTFRDHWFALV 138
    PRPALVFADYWETLY 139
    PAFSRFWSDLSAGAH 140
    PAFSRFWSKLSAGAH 141
    PXFXDYWXXL 142
    QETFSDLWKLLP 143
    QPTFSDLWKLLP 144
    QETFSDYWKLLP 145
    QPTFSDYWKLLP 146
  • [0105]
    TABLE 14
    Calmodulin antagonist peptide sequences
    Sequence/structure SEQ ID NO:
    SCVKWGKKEFCGS 164
    SCWKYWGKECGS 165
    SCYEWGKLRWCGS 166
    SCLRWGKWSNCGS 167
    SCWRWGKYQICGS 168
    SCVSWGALKLCGS 169
    SCIRWGQNTFCGS 170
    SCWQWGNLKICGS 171
    SCVRWGQLSICGS 172
    LKKFNARRKLKGAILTTMLAK 173
    RRWKKNFIAVSAANRFKK 174
    RKWQKTGHAVRAIGRLSS 175
    INLKALAALAKKIL 176
    KIWSILAPLGTTLVKLVA 177
    LKKLLKLLKKLLKL 178
    LKWKKLLKLLKKLLKKLL 179
    AEWPSLTEIKTLSHFSV 180
    AEWPSPTRVISTTYFGS 181
    AELAHWPPVKTVLRSFT 182
    AEGSWLQLLNLMKQMNN 183
    AEWPSLTEIK 184
  • [0106]
    TABLE 15
    Mast cell antagonists/Mast cell protease inhibitor
    peptide sequences
    Sequence/structure SEQ ID NO:
    SGSGVLKRPLPILPVTR 272
    RWLSSRPLPPLPLPPRT 273
    GSGSYDTLALPSLPLHPMSS 274
    GSGSYDTRALPSLPLHPMSS 275
    GSGSSGVTMYPKLPPHWSMA 276
    GSGSSGVRMYPKLPPHWSMA 277
    GSGSSSMRMVPTIPGSAKHG 278
    RNR NR
    QT NR
    RQK NR
    NRQ NR
    RQK NR
    RNRQKT 436
    RNRQ 437
    RNRQK 438
    NRQKT 439
    RQKT 440
  • [0107]
    TABLE 16
    SH3 antagonist peptide sequences
    Sequence/structure SEQ ID NO:
    RPLPPLP 282
    RELPPLP 283
    SPLPPLP 284
    GPLPPLP 285
    RPLPIPP 286
    RPLPIPP 287
    RRLPPTP 288
    RQLPPTP 289
    RPLPSRP 290
    RPLPTRP 291
    SRLPPLP 292
    RALPSPP 293
    RRLPRTP 294
    RPVPPIT 295
    ILAPPVP 296
    RPLPMLP 297
    RPLPILP 298
    RPLPSLP 299
    RPLPSLP 300
    RPLPMIP 301
    RPLPLIP 302
    RPLPPTP 303
    RSLPPLP 304
    RPQPPPP 305
    RQLPIPP 306
    XXXRPLPPLPXP 307
    XXXRPLPPIPXX 308
    XXXRPLPPLPXX 309
    RXXRPLPPLPXP 310
    RXXRPLPPLPPP 311
    PPPYPPPPIPXX 312
    PPPYPPPPVPXX 313
    LXXRPLPXΨP 314
    ΨPXXRPLPXLP 315
    PPXΘPPPΨP 316
    +PPΨPXKPXWL 317
    RPXΨPΨR+SXP 318
    PPVPPRPXXTL 319
    ΨPΨLPΨK 320
    +ΘDXPLPXLP 321
  • [0108]
    TABLE 17
    Somatostatin or cortistatin mimetic peptide sequences
    Sequence/structure SEQ ID NO:
    X1-X2-Asn-Phe-Phe-Trp-Lys-Thr-Phe-X3-Ser-X4 473
    Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 474
    Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 475
    Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 476
    Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 477
    Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 478
    Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 479
    Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 480
    Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 481
    Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 482
    Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 483
    Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 484
    Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 485
    Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 486
    Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 487
    Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 488
    Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 489
    Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 490
    Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 491
    Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 492
    Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 493
    Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 494
    Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 495
    Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 496
    Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 497
  • [0109]
    TABLE 18
    UKR antagonist peptide sequences
    Sequence/structure SEQ ID NO:
    AEPMPHSLNFSQYLWYT 196
    AEHTYSSLWDTYSPLAF 197
    AELDLWMRHYPLSFSNR 198
    AESSLWTRYAWPSMPSY 199
    AEWHPGLSFGSYLWSKT 200
    AEPALLNWSFFFNPGLH 201
    AEWSFYNLHLPEPQTIF 202
    AEPLDLWSLYSLPPLAM 203
    AEPTLWQLYQFPLRLSG 204
    AEISFSELMWLRSTPAF 205
    AELSEADLWTTWFGMGS 206
    AESSLWRIFSPSALMMS 207
    AESLPTLTSILWGKESV 208
    AETLFMDLWHDKHILLT 209
    AEILNFPLWHEPLWSTE 210
    AESQTGTLNTLFWNTLR 211
    AEPVYQYELDSYLRSYY 430
    AELDLSTFYDIQYLLRT 431
    AEFFKLGPNGYVYLHSA 432
    FKLXXXGYVYL 433
    AESTYHHLSLGYMYTLN 434
    YHXLXXGYMYT 435
  • [0110]
    TABLE 19
    Macrophage and/or T-cell
    inhibiting peptide sequences
    Sequence/structure SEQ ID NO:
    Xaa-Yaa-Arg NR
    Arg-Yaa-Xaa NR
    Xaa-Arg-Yaa NR
    Yaa-Arg-Xaa NR
    Ala-Arg NR
    Arg-Arg NR
    Asn-Arg NR
    Asp-Arg NR
    Cys-Arg NR
    Gln-Arg NR
    Glu-Arg NR
    Gly-Arg NR
    His-arg NR
    Ile-Arg NR
    Leu-Arg NR
    Lys-Arg NR
    Met-Arg NR
    Phe-Arg NR
    Ser-Arg NR
    Thr-Arg NR
    Trp-Arg NR
    Tyr-Arg NR
    Val-Arg NR
    Ala-Glu-Arg NR
    Arg-Glu-Arg NR
    Asn-Glu-Arg NR
    Asp-Glu-Arg NR
    Cys-Glu-Arg NR
    Gln-Glu-Arg NR
    Glu-Glu-Arg NR
    Gly-Glu-Arg NR
    His-Glu-Arg NR
    IIe-Glu-Arg NR
    Leu-Glu-Arg NR
    Lys-Glu-Arg NR
    Met-Glu-Arg NR
    Phe-Glu-Arg NR
    Pro-Glu-Arg NR
    Ser-Glu-Arg NR
    Thr-Glu-Arg NR
    Trp-Glu-Arg NR
    Tyr-Glu-Arg NR
    Val-Glu-Arg NR
    Arg-Ala NR
    Arg-Asp NR
    Arg-Cys NR
    Arg-Gln NR
    Arg-Glu NR
    Arg-Gly NR
    Arg-His NR
    Arg-Ile NR
    Arg-Leu NR
    Arg-Lys NR
    Arg-Met NR
    Arg-Phe NR
    Arg-Pro NR
    Arg-Ser NR
    Arg-Thr NR
    Arg-Trp NR
    Arg-Tyr NR
    Arg-Val NR
    Arg-Glu-Ala NR
    Arg-Glu-Asn NR
    Arg-Glu-Asp NR
    Arg-Glu-Cys NR
    Arg-Glu-Gln NR
    Arg-Glu-Glu NR
    Arg-Glu-Gly NR
    Arg-Glu-His NR
    Arg-Glu-Ile NR
    Arg-Glu-Leu NR
    Arg-Glu-Lys NR
    Arg-Glu-Met NR
    Arg-Glu-Phe NR
    Arg-Glu-Pro NR
    Arg-Glu-Ser NR
    Arg-Glu-Thr NR
    Arg-Glu-Trp NR
    Arg-Glu-Tyr NR
    Arg-Glu-Val NR
    Ala-Arg-Glu NR
    Arg-Arg-Glu NR
    Asn-Arg-Glu NR
    Asp-Arg-Glu NR
    Cys-Arg-Glu NR
    Gln-Arg-Glu NR
    Glu-Arg-Glu NR
    Gly-Arg-Glu NR
    His-Arg-Glu NR
    Ile-Arg-Glu NR
    Leu-Arg-Glu NR
    Lys-Arg-Glu NR
    Met-Arg-Glu NR
    Phe-Arg-Glu NR
    Pro-Arg-Glu NR
    Ser-Arg-Glu NR
    Thr-Arg-Glu NR
    Trp-Arg-Glu NR
    Tyr-Arg-Glu NR
    Val-Arg-Glu NR
    Glu-Arg-Ala NR
    Glu-Arg-Arg NR
    Glu-Arg-Asn NR
    Glu-Arg-Asp NR
    Glu-Arg-Cys NR
    Glu-Arg-Gln NR
    Glu-Arg-Gly NR
    Glu-Arg-His NR
    Glu-Arg-Ile NR
    Glu-Arg-Leu NR
    Glu-Arg-Lys NR
    Glu-Arg-Met NR
    Glu-Arg-Phe NR
    Glu-Arg-Pro NR
    Glu-Arg-Ser NR
    Glu-Arg-Thr NR
    Glu-Arg-Trp NR
    Glu-Arg-Tyr NR
    Glu-Arg-Val NR
  • [0111]
    TABLE 20
    Additional Exemplary Pharmacologically Active Peptides
    SEQ ID
    Sequence/structure NO: Activity
    VEPNCDIHVMWEWECFERL 1027 VEGF-antagonist
    GERWCFDGPLTWVCGEES 1084 VEGF-antagonist
    RGWVEICVADDNGMCVTEAQ 1085 VEGF-antagonist
    GWDECDVARMWEWECFAGV 1086 VEGF-antagonist
    GERWCFDGPRAWVCGWEI 501 VEGF-antagonist
    EELWCFDGPRAWVCGYVK 502 VEGF-antagonist
    RGWVEICAADDYGRCLTEAQ 1031 VEGF-antagonist
    RGWVEICESDVWGRCL 1087 VEGF-antagonist
    RGWVEICESDVWGRCL 1088 VEGF-antagonist
    GGNECDIARMWEWECFERL 1089 VEGF-antagonist
    RGWVEICAADDYGRCL 1090 VEGF-antagonist
    CTTHWGFTLC 1028 MMP inhibitor
    CLRSGXGC 1091 MMP inhibitor
    CXXHWGFXXC 1092 MMP inhibitor
    CXPXC 1093 MMP inhibitor
    CRRHWGFEFC 1094 MMP inhibitor
    STTHWGFTLS 1095 MMP inhibitor
    CSLHWGFWWC 1096 CTLA4-mimetic
    GFVCSGIFAVGVGRC 125 CTLA4-mimetic
    APGVRLGCAVLGRYC 126 CTLA4-mimetic
    LLGRMK 105 Antiviral (HBV)
    ICVVQDWGHHRCTAGHMANLTSHASAI 127 C3b antagonist
    ICVVQDWGHHRCT 128 C3b antagonist
    CVVQDWGHHAC 129 C3b antagonist
    STGGFDDVYDWARGVSSALTTTLVATR 185 Vinculin-binding
    STGGFDDVYDWARRVSSALTTTLVATR 186 Vinculin-binding
    SRGVNFSEWLYDMSAAMKEASNVFPSRRSR 187 Vinculin-binding
    SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR 188 Vinculin-binding
    SSPSLYTQFLVNYESAATRIQDLLIASRPSR 189 Vinculin-binding
    SSTGWVDLLGALQRAADATRTSIPPSLQNSR 190 Vinculin-binding
    DVYTKKELIECARRVSEK 191 Vinculin-binding
    EKGSYYPGSGIAQFHIDYNNVS 192 C4BP-binding
    SGIAQFHIDYNNVSSAEGWHVN 193 C4BP-binding
    LVTVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVN 194 C4BP-binding
    SGIAQFHIDYNNVS 195 C4BP-binding
    LLGRMK 279 anti-HBV
    ALLGRMKG 280 anti-HBV
    LDPAFR 281 anti-HBV
    CXXRGDC 322 Inhibition of platelet
    aggregation
    RPLPPLP 323 Src antagonist
    PPVPPR 324 Src antagonist
    XFXDXWXXLXX 325 Anti-cancer
    (particularly for
    sarcomas)
    KACRRLFGPVDSEQLSRDCD 326 p16-mimetic
    RERWNFDFVTETPLEGDFAW 327 p16-mimetic
    KRRQTSMTDFYHSKRRLIFS 328 p16-mimetic
    TSMTDFYHSKRRLIFSKRKP 329 p16-mimetic
    RRLIF 330 p16-mimetic
    KRRQTSATDFYHSKRRLIFSRQIKIWFQNRRMKWKK 331 p16-mimetic
    KRRLIFSKRQIKIWFQNRRMKWKK 332 p16-mimetic
    Asn Gln Gly Arg His Phe Cys Gly Gly Ala Leu Ile His Ala 498 CAP37 mimetic/LPS
    Arg Phe Val Met Thr Ala Ala Ser Cys Phe Gln binding
    Arg His Phe Cys Gly Gly Ala Leu Ile His Ala Arg Phe Val 499 CAP37 mimetic/LPS
    Met Thr Ala Ala Ser Cys binding
    Gly Thr Arg Cys Gln Val Ala Gly Trp Gly Ser Gln Arg Ser 500 CAP37 mimetic/LPS
    Gly Gly Arg Leu Ser Arg Phe Pro Arg Phe Val Asn Val binding
    WHWRHRIPLQLAAGR 1097 carbohydrate (GD1
    alpha) mimetic
    LKTPRV 1098 β2GPI Ab binding
    NTLKTPRV 1099 β2GPI Ab binding
    NTLKTPRVGGC 1100 β2GPI Ab binding
    KDKATF 1101 β2GPI Ab binding
    KDKATFGCHD 1102 β2GPI Ab binding
    KDKATFGCHDGC 1103 β2GPI Ab binding
    TLRVYK 1104 β2GPI Ab binding
    ATLRVYKGG 1105 β2GPI Ab binding
    CATLRVYKGG 1106 β2GPI Ab binding
    INLKALAALAKKIL 1107 Membrane-
    transporting
    GWT NR Membrane-
    transporting
    GWTLNSAGYLLG 1108 Membrane-
    transporting
    GWTLNSAGYLLGKINLKALAALAKKIL 1109 Membrane-
    transporting
  • The present invention is also particularly useful with peptides having activity in treatment of: [0112]
  • cancer, wherein the peptide is a VEGF-mimetic or a VEGF receptor antagonist, a HER2 agonist or antagonist, a CD20 antagonist and the like; [0113]
  • asthma, wherein the protein of interest is a CKR3 antagonist, an IL-5 receptor antagonist, and the like; [0114]
  • thrombosis, wherein the protein of interest is a GPIIb antagonist, a GPIIIa antagonist, and the like; [0115]
  • autoimmune diseases and other conditions involving immune modulation, wherein the protein of interest is an IL-2 receptor antagonist, a CD40 agonist or antagonist, a CD40L agonist or antagonist, a thymopoietin mimetic and the like. [0116]
  • Vehicles. This invention requires the presence of at least one vehicle (F[0117] 1, F2) attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the amino acid residues. Multiple vehicles may also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or a sidechain.
  • An Fc domain is the preferred vehicle. The Fc domain may be fused to the N or C termini of the peptides or at both the N and C termini. For the TPO-mimetic peptides, molecules having the Fc domain fused to the N terminus of the peptide portion of the molecule are more bioactive than other such fusions, so fusion to the N terminus is preferred. As noted above, Fc variants are suitable vehicles within the scope of this invention. A native Fc may be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478. [0118]
  • In such Fc variants, one may remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants may be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which: [0119]
  • 1. Sites involved in disulfide bond formation are removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, one may truncate the N-terminal 20-amino acid segment of SEQ ID NO: 2 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently. [0120]
  • 2. A native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc, which may be recognized by a digestive enzyme in [0121] E. coli such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 2 (FIG. 4) is one such Fc variant.
  • 3. A portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5. [0122]
  • 4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine). [0123]
  • 5. Sites involved in interaction with complement, such as the C1q binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so may be avoided with such an Fc variant. [0124]
  • 6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc may have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so may be removed. [0125]
  • 7. The ADCC site is removed. ADCC sites are known in the art; see, for example, [0126] Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed.
  • 8. When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art. [0127]
  • Preferred Fc variants include the following. In SEQ ID NO: 2 (FIG. 4) the leucine at position 15 may be substituted with glutamate; the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, one or more tyrosine residues can be replaced by phenyalanine residues. [0128]
  • An alternative vehicle would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “vehicle” and are within the scope of this invention. Such vehicles should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization). [0129]
  • As noted above, polymer vehicles may also be used for F[0130] 1 and F2. Various means for attaching chemical moieties useful as vehicles are currently available, see, e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins.
  • A preferred polymer vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton (“kD”) to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kDa to about 10 kDa. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group). [0131]
  • A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis (see, for example, FIGS. 5 and 6 and the accompanying text herein). The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry. [0132]
  • Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention. [0133]
  • Linkers. Any “linker” group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. The linker is preferably made up of amino acids linked together by peptide bonds. Thus, in preferred embodiments, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly)[0134] 4, (Gly)5), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:
    (Gly)3Lys(Gly)4; (SEQ ID NO: 333)
    (Gly)3AsnGlySer(Gly)2; (SEQ ID NO: 334)
    (Gly)3Cys(Gly)4; and (SEQ ID NO: 335)
    GlyProAsnGlyGly. (SEQ ID NO: 336)
  • To explain the above nomenclature, for example, (Gly)[0135] 3Lys(Gly)4 means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are also preferred. The linkers shown here are exemplary; linkers within the scope of this invention may be much longer and may include other residues.
  • Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH[0136] 2)s—C(O)—, wherein s=2-20 could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker,
    Figure US20040071712A1-20040415-C00013
  • wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be altered to form derivatives in the same manner as described above. [0137]
  • Derivatives. The inventors also contemplate derivatizing the peptide and/or vehicle portion of the compounds. Such derivatives may improve the solubility, absorption, biological half life, and the like of the compounds. The moieties may alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. Exemplary derivatives include compounds in which: [0138]
  • 1. The compound or some portion thereof is cyclic. For example, the peptide portion may be modified to contain two or more Cys residues (e.g., in the linker), which could cyclize by disulfide bond formation. For citations to references on preparation of cyclized derivatives, see Table 2. [0139]
  • 2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion may be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound may also be cross-linked through its C-terminus, as in the molecule shown below. [0140]
    Figure US20040071712A1-20040415-C00014
  • 3. [0141]
  • 4. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages are —CH[0142] 2-carbamate [—CH2—OC(O)NR—], phosphonate, —CH2-sulfonamide [—CH2—S(O)2NR—], urea [—NHC(O)NH—], —CH2-secondary amine, and alkylated peptide [—C(O)NR6— wherein R6 is lower alkyl].
  • 5. The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR[0143] 1 (other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R′ are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo.
  • 6. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. For example, one may use methods described in the art to add (NH—CH[0144] 2—CH2—NH2)2 to compounds of this invention having any of SEQ ID NOS: 504 to 508 at the C-terminus. Likewise, one may use methods described in the art to add —NH2 to compounds of this invention having any of SEQ ID NOS: 924 to 955, 963 to 972, 1005 to 1013, or 1018 to 1023 at the C-terminus. Exemplary C-terminal derivative groups include, for example, —C(O)R2 wherein R2 is lower alkoxy or —NR3R4 wherein R3 and R4 are independently hydrogen or C1-C8 alkyl (preferably C1-C4 alkyl).
  • 7. A disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), [0145] J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.
  • 8. One or more individual amino acid residues is modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below. [0146]
  • Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. [0147]
  • Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. [0148]
  • Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. [0149]
  • Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R[0150] 1-N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention. [0151]
  • Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), [0152] J. Med. Chem. 39: 3814-9.
  • Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization. [0153]
  • Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art. [0154]
  • Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, [0155] Proteins: Structure and Molecule Properties (W. H. Freeman & Co., San Francisco), pp. 79-86 (1983).
  • Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be -changed to codons more compatible with the chosen host cell. For [0156] E. coli, which is the preferred host cell, optimized codons are known in the art.
  • Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes. [0157]
  • Methods of Making [0158]
  • The compounds of this invention largely may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used. [0159]
  • The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation. [0160]
  • The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art. [0161]
  • Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as [0162] E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.
  • Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art. [0163]
  • The compounds may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), [0164] Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.)2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides.
  • Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques. [0165]
  • Uses of the Compounds [0166]
  • In general. The compounds of this invention have pharmacologic activity resulting from their ability to bind to proteins of interest as agonists, mimetics or antagonists of the native ligands of such proteins of interest. The utility of specific compounds is shown in Table 2. The activity of these compounds can be measured by assays known in the art. For the TPO-mimetic and EPO-mimetic compounds, in vivo assays are further described in the Examples section herein. [0167]
  • In addition to therapeutic uses, the compounds of the present invention are useful in diagnosing diseases characterized by dysfunction of their associated protein of interest. In one embodiment, a method of detecting in a biological sample a protein of interest (e.g., a receptor) that is capable of being activated comprising the steps of: (a) contacting the sample with a compound of this invention; and (b) detecting activation of the protein of interest by the compound. The biological samples include tissue specimens, intact cells, or extracts thereof. The compounds of this invention may be used as part of a diagnostic kit to detect the presence of their associated proteins of interest in a biological sample. Such kits employ the compounds of the invention having an attached label to allow for detection. The compounds are useful for identifying normal or abnormal proteins of interest. For the EPO-mimetic compounds, for example, presence of abnormal protein of interest in a biological sample may be indicative of such disorders as Diamond Blackfan anemia, where it is believed that the EPO receptor is dysfunctional. [0168]
  • Therapeutic uses of EPO-mimetic compounds. The EPO-mimetic compounds of the invention are useful for treating disorders characterized by low red blood cell levels. Included in the invention are methods of modulating the endogenous activity of an EPO receptor in a mammal, preferably methods of increasing the activity of an EPO receptor. In general, any condition treatable by erythropoietin, such as anemia, may also be treated by the EPO-mimetic compounds of the invention. These compounds are administered by an amount and route of delivery that is appropriate for the nature and severity of the condition being treated and may be ascertained by one skilled in the art. Preferably, administration is by injection, either subcutaneous, intramuscular, or intravenous. [0169]
  • Therapeutic uses of TPO-mimetic compounds. For the TPO-mimetic compounds, one can utilize such standard assays as those described in WO95/26746 entitled “Compositions and Methods for Stimulating Megakaryocyte Growth and Differentiation”. In vivo assays also appear in the Examples hereinafter. [0170]
  • The conditions to be treated are generally those that involve an existing megakaryocyte/platelet deficiency or an expected megakaryocyte/platelet deficiency (e.g., because of planned surgery or platelet donation). Such conditions will usually be the result of a deficiency (temporary or permanent) of active Mpl ligand in vivo. The generic term for platelet deficiency is thrombocytopenia, and hence the methods and compositions of the present invention are generally available for treating thrombocytopenia in patients in need thereof. Thrombocytopenia (platelet deficiencies) may be present for various reasons, including chemotherapy and other therapy with a variety of drugs, radiation therapy, surgery, accidental blood loss, and other specific disease conditions. Exemplary specific disease conditions that involve thrombocytopenia and may be treated in accordance with this invention are: aplastic anemia, idiopathic thrombocy openia, metastatic tumors which result in thrombocytopenia, systemic lupus erythematosus, splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic acid deficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT). Certain wound healing disorders might also benefit from an increase in platelet numbers. [0171]
  • With regard to anticipated platelet deficiencies, e.g., due to future surgery, a compound of the present invention could be administered several days to several hours prior to the need for platelets. With regard to acute situations, e.g., accidental and massive blood loss, a compound of this invention could be administered along with blood or purified platelets. [0172]
  • The TPO-mimetic compounds of this invention may also be useful in stimulating certain cell types other than megakaryocytes if such cells are found to express Mpl receptor. Conditions associated with such cells that express the Mpl receptor, which are responsive to stimulation by the Mpl ligand, are also within the scope of this invention. [0173]
  • The TPO-mimetic compounds of this invention may be used in any situation in which production of platelets or platelet precursor cells is desired, or in which stimulation of the c-Mpl receptor is desired. Thus, for example, the compounds of this invention may be used to treat any condition in a mammal wherein there is a need of platelets, megakaryocytes, and the like. Such conditions are described in detail in the following exemplary sources: WO95/26746; WO95/21919; WO95/18858; WO95/21920 and are incorporated herein. [0174]
  • The TPO-mimetic compounds of this invention may also be useful in maintaining the viability or storage life of platelets and/or megakaryocytes and related cells. Accordingly, it could be useful to include an effective amount of one or more such compounds in a composition containing such cells. [0175]
  • The therapeutic methods, compositions and compounds of the present invention may also be employed, alone or in combination with other cytokines, soluble Mpl receptor, hematopoietic factors, interleukins, growth factors or antibodies in the treatment of disease states characterized by other symptoms as well as platelet deficiencies. It is anticipated that the inventive compound will prove useful in treating some forms of thrombocytopenia in combination with general stimulators of hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocytic stimulatory factors, i.e., meg-CSF, stem cell factor (SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or other molecules with megakaryocyte stimulating activity may also be employed with Mpl ligand. Additional exemplary cytokines or hematopoietic factors for such co-administration include IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, or IFN-gamma. It may further be useful to administer, either simultaneously or sequentially, an effective amount of a soluble mammalian Mpl receptor, which appears to have an effect of causing megakaryocytes to fragment into platelets once the megakaryocytes have reached mature form. Thus, administration of an inventive compound (to enhance the number of mature megakaryocytes) followed by administration of the soluble Mpl receptor (to inactivate the ligand and allow the mature megakaryocytes to produce platelets) is expected to be a particularly effective means of stimulating platelet production. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by conventional methods. [0176]
  • In cases where the inventive compounds are added to compositions of platelets and/or megakaryocytes and related cells, the amount to be included will generally be ascertained experimentally by techniques and assays known in the art. An exemplary range of amounts is 0.1 μg-1 mg inventive compound per 10[0177] 6 cells.
  • Pharmaceutical Compositions [0178]
  • In General. The present invention also provides methods of using pharmaceutical compositions of the inventive compounds. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal, transdermal or other forms of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of a compound of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations. [0179]
  • Oral dosage forms. Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89 of Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall, K., [0180] Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated by reference. In general, the formulation will include the inventive compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • Also specifically contemplated are oral dosage forms of the above inventive compounds. If necessary, the compounds may be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached vehicles in this invention may also be used for this purpose. [0181]
  • Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis, [0182] Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.
  • For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods”. [0183]
  • The compounds of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression. [0184]
  • Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents. [0185]
  • One may dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell. [0186]
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. [0187]
  • Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic. [0188]
  • An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. [0189]
  • Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate. [0190]
  • To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios. [0191]
  • Additives may also be included in the formulation to enhance uptake of the compound. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid. [0192]
  • Controlled release formulation may be desirable. The compound of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect. [0193]
  • Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid. [0194]
  • A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating. [0195]
  • Pulmonary delivery forms. Also contemplated herein is pulmonary delivery of the present protein (or derivatives thereof). The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., [0196] Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84:1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization of Proteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140: 3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).
  • Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass. [0197]
  • All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy. [0198]
  • The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung. [0199]
  • Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation. [0200]
  • Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. [0201]
  • Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol. [0202]
  • Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. [0203]
  • Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. [0204]
  • Nasal delivery forms. Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated. [0205]
  • Dosages. The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram. [0206]
  • Specific Preferred Embodiments [0207]
  • The inventors have determined preferred peptide sequences for molecules having many different kinds of activity. The inventors have further determined preferred structures of these preferred peptides combined with preferred linkers and vehicles. Preferred structures for these preferred peptides listed in Table 21 below. [0208]
    TABLE 21
    Preferred embodiments
    SEQ ID
    Sequence/structure NO: Activity
    F1-(G)5-IEGPTLRQWLAARA-(G)8-IEGPTLRQWLAARA 337 TPO-mimetic
    IEGPTLRQWLAARA-(G)8-IEGPTLRQWLAARA-(G)5-F1 338 TPO-mimetic
    F1-(G)5-IEGPTLRQWLAARA 1032 TPO-mimetic
    IEGPTLRQWLAARA-(G)5-F1 1033 TPO-mimetic
    F1-(G)5-GGTYSCHFGPLTWVCKPQGG-(G)4- 339 EPO-mimetic
    GGTYSCHFGPLTWVCKPQGG
    GGTYSCHFGPLTWVCKPQGG-(G)4- 340 EPO-mimetic
    GGTYSCHFGPLTWVCKPQGG-(G)5-F1
    GGTYSCHFGPLTWVCKPQGG-(G)5-F1 1034 EPO-mimetic
    F1-(G)5-DFLPHYKNTSLGHRP 1045 TNF-α inhibitor
    DFLPHYKNTSLGHRP-(G)5-F1 1046 TNF-α inhibitor
    F1-(G)5-FEWTPGYWQPYALPL 1047 IL-1 R antagonist
    FEWTPGYWQPYALPL-(G)5-F1 1048 IL-1 R antagonist
    F1-(G)5-VEPNCDIHVMWEWECFERL 1049 VEGF-antagonist
    VEPNCDIHVMWEWECFERL-(G)5-F1 1050 VEGF-antagonist
    F1-(G)5-CTTHWGFTLC 1051 MMP inhibitor
    CTTHWGFTLC-(G)5-F1 1052 MMP inhibitor
  • WORKING EXAMPLES
  • The compounds described above may be prepared as described below. These examples comprise preferred embodiments of the invention and are illustrative rather than limiting. [0209]
  • Example 1 TPO-Mimetics
  • The following example uses peptides identified by the numbers appearing in Table A hereinafter. [0210]
  • Preparation of peptide 19. Peptide 17b (12 mg) and MeO-PEG-SH 5000 (30 mg, 2 equiv.) were dissolved in 1 ml aqueous buffer (pH 8). The mixture was incubated at RT for about 30 minutes and the reaction was checked by analytical HPLC, which showed a >80% completion of the reaction. The pegylated material was isolated by preparative HPLC. [0211]
  • Preparation of peptide 20. Peptide 18 (14 mg) and MeO-PEG-maleimide (25 mg) were dissolved in about 1.5 ml aqueous buffer (pH 8). The mixture was incubated at RT for about 30 minutes, at which time about 70% transformation was complete as monitored with analytical HPLC by applying an aliquot of sample to the HPLC column. The pegylated material was purified by preparative HPLC. [0212]
  • Bioactivity assay. The TPO in vitro bioassay is a mitogenic assay utilizing an IL-3 dependent clone of murine 32D cells that have been transfected with human mpl receptor. This assay is described in greater detail in WO 95/26746. Cells are maintained in MEM medium containing 10% Fetal Clone II and 1 ng/ml mIL-3. Prior to sample addition, cells are prepared by rinsing twice with growth medium lacking mIL-3. An extended twelve point TPO standard curve is prepared, ranging from 33 to 39 μg/ml. Four dilutions, estimated to fall within the linear portion of the standard curve, (100 to 125 μg/ml), are prepared for each sample and run in triplicate. A volume of 100 μl of each dilution of sample or standard is added to appropriate wells of a 96 well microtiter plate -containing 10,000 cells/well. After forty-four hours at 37° C. and 10% CO[0213] 2, MTS (a tetrazolium compound which is bioreduced by cells to a formazan) is added to each well. Approximately six hours later, the optical density is read on a plate reader at 490 nm. A dose response curve (log TPO concentration vs. O.D.—Background) is generated and linear regression analysis of points which fall in the linear portion of the standard curve is performed. Concentrations of unknown test samples are determined using the resulting linear equation and a correction for the dilution factor.
  • TMP tandem repeats with polyglycine linkers. Our design of sequentially linked TMP repeats was based on the assumption that a dimeric form of TMP was required for its effective interaction with c-Mpl (the TPO receptor) and that depending on how they were wound up against each other in the receptor context, the two TMP molecules could be tethered together in the C- to N-terminus configuration in a way that would not perturb the global dimeric conformation. Clearly, the success of the design of tandem linked repeats depends on proper selection of the length and composition of the linker that joins the C- and N-termini of the two sequentially aligned TMP monomers. Since no structural information of the TMP bound to c-Mpl was available, a series of repeated peptides with linkers composed of 0 to 10 and 14 glycine residues (Table A) were synthesized. Glycine was chosen because of its simplicity and flexibility, based on the rationale that a flexible polyglycine peptide chain might allow for the free folding of the two tethered TMP repeats into the required conformation, while other amino acid sequences may adopt undesired secondary structures whose rigidity might disrupt the correct packing of the repeated peptide in the receptor context. [0214]
  • The resulting peptides are readily accessible by conventional solid phase peptide synthesis methods (Merrifield (1963), [0215] J. Amer. Chem. Soc. 85: 2149) with either Fmoc or t-Boc chemistry. Unlike the synthesis of the C-terminally linked parallel dimer which required the use of an orthogonally protected lysine residue as the initial branch point to build the two peptide chains in a pseudosymmetrical way (Cwirla et al. (1997), Science 276: 1696-9), the synthesis of these tandem repeats was a straightforward, stepwise assembly of the continuous peptide chains from the C- to N-terminus. Since dimerization of TMP had a more dramatic effect on the proliferative activity than binding affinity as shown for the C-terminal dimer (Cwirla et al. (1997)), the synthetic peptides were tested directly for biological activity in a TPO-dependent cell-proliferation assay using an IL-3 dependent clone of murine 32D cells transfected with the full-length c-Mpl (Palacios et al., Cell 41:727 (1985)). As the test results showed, all the polyglycine linked tandem repeats demonstrated >1000 fold increases in potency as compared to the monomer, and were even more potent than the C-terminal dimer in this cell proliferation assay. The absolute activity of the C-terminal dimer in our assay was lower than that of the native TPO protein, which is different from the previously reported findings in which the C-terminal dimer was found to be as active as the natural ligand (Cwirla et al. (1997)). This might be due to differences in the conditions used in the two assays. Nevertheless, the difference in activity between tandem (C terminal of first monomer linked to N terminal of second monomer) and C-terminal (C terminal of first monomer linked to C terminal of second monomer; also referred to as parallel) dimers in the same assay clearly demonstrated the superiority of tandem repeat strategy over parallel peptide dimerization. It is interesting to note that a wide range of length is tolerated by the linker. The optimal linker between tandem peptides with the selected TMP monomers apparently is composed of 8 glycines.
  • Other tandem repeats. Subsequent to this first series of TMP tandem repeats, several other molecules were designed either with different linkers or containing modifications within the monomer itself. [0216]
  • The first of these molecules, peptide 13, has a linker composed of GPNG, a sequence known to have a high propensity to form a β-turn-type secondary structure. Although still about 100-fold more potent than the monomer, this peptide was found to be >10-fold less active than the equivalent GGGG-linked analog. Thus, introduction of a relatively rigid β-turn at the linker region seemed to have caused a slight distortion of the optimal agonist conformation in this short linker form. [0217]
  • The Trp9 in the TMP sequence is a highly conserved residue among the active peptides isolated from random peptide libraries. There is also a highly conserved Trp in the consensus sequences of EPO mimetic peptides and this Trp residue was found to be involved in the formation of a hydrophobic core between the two EMPs and contributed to hydrophobic interactions with the EPO receptor. Livnah et al. (1996), [0218] Science 273: 464-71). By analogy, the Trp9 residue in TMP might have a similar function in dimerization of the peptide ligand, and as an attempt to modulate and estimate the effects of noncovalent hydrophobic forces exerted by the two indole rings, several analogs were made resulting from mutations at the Trp. So in peptide 14, the Trp residue was replaced in each of the two TMP monomers with a Cys, and an intramolecular disulfide bond was formed between the two cysteines by oxidation which was envisioned to mimic the hydrophobic interactions between the two Trp residues in peptide dimerization. Peptide 15 is the reduced form of peptide 14. In peptide 16, the two Trp residues were replaced by Ala. As the assay data show, all three analogs were inactive. These data further demonstrated that Trp is critical for the activity of the TPO mimetic peptide, not just for dimer formation.
  • The next two peptides (peptide 17a, and 18) each contain in their 8-amino acid linker a Lys or Cys residue. These two compounds are precursors to the two PEGylated peptides (peptide 19 and 20) in which the side chain of the Lys or Cys is modified by a PEG moiety. A PEG moiety was introduced at the middle of a relatively long linker, so that the large PEG component (5 kDa) is far enough away from the critical binding sites in the peptide molecule. PEG is a known biocompatible polymer which is increasingly used as a covalent modifier to improve the pharmacokinetic profiles of peptide- and protein-based therapeutics. [0219]
  • A modular, solution-based method was devised for convenient PEGylation of synthetic or recombinant peptides. The method is based on the now well established chemoselective ligation strategy which utilizes the specific reaction between a pair of mutually reactive functionalities. So, for pegylated peptide 19, the lysine side chain was preactivated with a bromoacetyl group to give peptide 17b to accommodate reaction with a thiol-derivatized PEG. To do that, an orthogonal protecting group, Dde, was employed for the protection of the lysine ε-amine. Once the whole peptide chain was assembled, the N-terminal amine was reprotected with t-Boc. Dde was then removed to allow for the bromoacetylation. This strategy gave a high quality crude peptide which was easily purified using conventional reverse phase HPLC. Ligation of the peptide with the thiol-modified PEG took place in aqueous buffer at pH 8 and the reaction completed within 30 minutes. MALDI-MS analysis of the purified, pegylated material revealed a characteristic, bell-shaped spectrum with an increment of 44 Da between the adjacent peaks. For PEG-peptide 20, a cysteine residue was placed in the linker region and its side chain thiol group would serve as an attachment site for a maleimide-containing PEG. [0220]
  • Similar conditions were used for the pegylation of this peptide. As the assay data revealed, these two pegylated peptides had even higher in vitro bioactivity as compared to their unpegylated counterparts. [0221]
  • Peptide 21 has in its 8-amino acid linker a potential glycosylation motif, NGS. Since our exemplary tandem repeats are made up of natural amino acids linked by peptide bonds, expression of such a molecule in an appropriate eukaryotic cell system should produce a glycopeptide with the carbohydrate moiety added on the side chain carboxyamide of Asn. Glycosylation is a common post-translational modification process which can have many positive impacts on the biological activity of a given protein by increasing its aqueous solubility and in vivo stability. As the assay data show, incorporation of this glycosylation motif into the linker maintained high bioactivity. The synthetic precursor of the potential glycopeptide had in effect an activity comparable to that of the -(G)[0222] 8-linked analog. Once glycosylated, this peptide is expected to have the same order of activity as the pegylated peptides, because of the similar chemophysical properties exhibited by a PEG and a carbohydrate moiety.
  • The last peptide is a dimer of a tandem repeat. It was prepared by oxidizing peptide 18, which formed an intermolecular disulfide bond between the two cysteine residues located at the linker. This peptide was designed to address the possibility that TMP was active as a tetramer. The assay data showed that this peptide was not more active than an average tandem repeat on an adjusted molar basis, which indirectly supports the idea that the active form of TMP is indeed a dimer, otherwise dimerization of a tandem repeat would have a further impact on the bioactivity. [0223]
  • In order to confirm the in vitro data in animals, one pegylated TMP tandem repeat (compound 20 in Table A) was delivered subcutaneously to normal mice via osmotic pumps. Time and dose-dependent increases were seen in platelet numbers for the duration of treatment. Peak platelet levels over 4-fold baseline were seen on day 8. A dose of 10 μg/kg/day of the pegylated TMP repeat produced a similar response to rHuMGDF (non-pegylated) at 100 μg/kg/day delivered by the same route. [0224]
    TABLE A
    TPO-mimetic Peptides
    Peptide SEQ ID Relative
    No. Compound NO: Potency
    TPO ++++
    TMP monomer 13 +
    TMP C-C dimer +++−
    TMP-(G)n-TMP:
     1 n = 0 341 ++++−
     2 n = 1 342 ++++
     3 n = 2 343 ++++
     4 n = 3 344 ++++
     5 n = 4 345 ++++
     6 n = 5 346 ++++
     7 n = 6 347 ++++
     8 n = 7 348 ++++
     9 n = 8 349 ++++−
    10 n = 9 350 ++++
    11 n = 10 351 ++++
    12 n = 14 352 ++++
    13 TMP-GPNG-TMP 353 +++
    14
    Figure US20040071712A1-20040415-C00015
    354
    15 IEGPTLRQWCLAARA-GGGGGGGG- 355
    IEGPTLRQCLAARA-(linear)
    16 IEGPTLRQALAARA-GGGGGGGG- 356
    IEGPTLRQALAARA
    17a TMP-GGGKGGGG-TMP 357 ++++
    17b TMP-GGGK(BrAc)GGGG-TMP 358 ND
    18 TMNP-GGGCGGGG-TMP 359 ++++
    19 TMP-GGGK(PEG)GGGG-TMP 360 +++++
    20 TMP-GGGC(PEG)GGGG-TMP 361 +++++
    21 TMP-GGGN*GSGG-TMP 362 ++++
    22 TMP-GGGCGGGG-TMP 363
        | ++++
    TMP-GGGCGGGG-TMP 363
  • Discussion. It is well accepted that MGDF acts in a way similar to hGH, i.e., one molecule of the protein ligand binds two molecules of the receptor for its activation. Wells et al.(1996), [0225] Ann. Rev. Biochem. 65: 609-34. Now, this interaction is mimicked by the action of a much smaller peptide, TMP. However, the present studies suggest that this mimicry requires the concerted action of two TMP molecules, as covalent dimerization of TMP in either a C—C parallel or C—N sequential fashion increased the in vitro biological potency of the original monomer by a factor of greater than 103. The relatively low biopotency of the monomer is probably due to inefficient formation of the noncovalent dimer. A preformed covalent repeat has the ability to eliminate the entropy barrier for the formation of a noncovalent dimer which is exclusively driven by weak, noncovalent interactions between two molecules of the small, 14-residue peptide.
  • It is intriguing that this tandem repeat approach had a similar effect on enhancing bioactivity as the reported C—C dimerization is intriguing. These two strategies brought about two very different molecular configurations. The C—C dimer is a quasi-symmetrical molecule, while the tandem repeats have no such symmetry in their linear structures. Despite this difference in their primary structures, these two types of molecules appeared able to fold effectively into a similar biologically active conformation and cause the dimerization and activation of c-Mpl. These experimental observations provide a number of insights into how the two TMP molecules may interact with one another in binding to c-Mpl. First, the two C-termini of the two bound TMP molecules must be in relatively close proximity with each other, as suggested by data on the C-terminal dimer. Second, the respective N- and C-termini of the two TMP molecules in the receptor complex must also be very closely aligned with each other, such that they can be directly tethered together with a single peptide bond to realize the near maximum activity-enhancing effect brought about by the tandem repeat strategy. Insertion of one or more (up to 14) glycine residues at the junction did not increase (or decrease) significantly the activity any further. This may be due to the fact that a flexible polyglycine peptide chain is able to loop out easily from the junction without causing any significant changes in the overall conformation. This flexibility seems to provide the freedom of orientation for the TMP peptide chains to fold into the required conformation in interacting with the receptor and validate it as a site of modification. Indirect evidence supporting this came from the study on peptide 13, in which a much more rigid b-turn-forming sequence as the linker apparently forced a deviation of the backbone alignment around the linker which might have resulted in a slight distortion of the optimal conformation, thus resulting in a moderate (10-fold) decrease in activity as compared with the analogous compound with a 4-Gly linker. Third, Trp9 in TMP plays a similar role as Trp13 in EMP, which is involved not only in peptide:peptide interaction for the formation of dimers but also is important for contributing hydrophobic forces in peptide:receptor interaction. Results obtained with the W to C mutant analog, peptide 14, suggest that a covalent disulfide linkage is not sufficient to approximate the hydrophobic interactions provided by the Trp pair and that, being a short linkage, it might bring the two TMP monomers too close, therefore perturbing the overall conformation of the optimal dimeric structure. [0226]
  • An analysis of the possible secondary structure of the TMP peptide can provide further understanding on the interaction between TMP and c-Mpl. This can be facilitated by making reference to the reported structure of the EPO mimetic peptide. Livnah et al. (1996), [0227] Science 273:464-75 The receptor-bound EMP has a b-hairpin structure with a b-turn formed by the highly consensus Gly-Pro-Leu-Thr at the center of its sequence. Instead of GPLT, TMP has a highly selected GPTL sequence which is likely to form a similar turn. However, this turn-like motif is located near the N-terminal part in TMP. Secondary structure prediction using Chau-Fasman method suggests that the C-terminal half of the peptide has a tendency to adopt a helical conformation. Together with the highly conserved Trp at position 9, this C-terminal helix may contribute to the stabilization of the dimeric structure. It is interesting to note that most of our tandem repeats are more potent than the C-terminal parallel dimer. Tandem repeats seem to give the molecule a better fit conformation than does the C—C parallel dimerization. The seemingly asymmetric feature of a tandem repeat might have brought it closer to the natural ligand which, as an asymmetric molecule, uses two different sites to bind two identical receptor molecules.
  • Introduction of a PEG moiety was envisaged to enhance the in vivo activity of the modified peptide by providing it a protection against proteolytic degradation and by slowing down its clearance through renal filtration. It was unexpected that pegylation could further increase the in vitro bioactivity of a tandem repeated TMP peptide in the cell-based proliferation assay. [0228]
  • Example 2 Fc-TMP Fusions
  • TMPs (and EMPs as described in Example 3) were expressed in either monomeric or dimeric form as either N-terminal or C-terminal fusions to the Fc region of human IgG1. In all cases, the expression construct utilized the luxPR promoter promoter in the plasmid expression vector pAMG21. [0229]
  • Fc-TMP. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of the TPO-mimetic peptide was constructed using standard PCR technology. Templates for PCR reactions were the pFc-A3 vector and a synthetic TMP gene. The synthetic gene was constructed from the 3 overlapping oligonucleotides (SEQ ID NOS: 364, 365, and 366, respectively) shown below: [0230]
    1842-97 AAA AAA GGA TCC TCG AGA TTA AGC ACG AGC
    AGC CAG CCA CTG ACG CAG AGT CGG ACC
    1842-98 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCG
    ACT CTG CGT
    1842-99 CAG TGG CTG GCT GCT CGT GCT TAA TCT CGA
    GGA TCC TTT TTT
  • These oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 367 and 368, respectively) shown below: [0231]
    AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT
    1 ---------+---------+---------+---------+---------+---------+ 60
                            CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA
    a K  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A  R  A
    TAATCTCGAGGATCCTTTTTT
    61 ---------+---------+-  81
    ATTAGAGCTCCTAGGAAAAAA
    a *
  • This duplex was amplified in a PCR reaction using 1842-98 and 1842-97 as the sense and antisense primers. [0232]
  • The Fc portion of the molecule was generated in a PCR reaction with pFc-A3 using the primers shown below (SEQ ID NOS: 369 and 370): [0233]
    1216-52 AAC ATA AGT ACC TGT AGG ATC G
    1830-51 TTCGATACCA CCACCTCCAC CTTTACCCGG AGACAGGGAG AGGCTCTTCTGC
  • The oligonucleotides 1830-51 and 1842-98 contain an overlap of 24 nucleotides, allowing the two genes to be fused together in the correct reading frame by combining the above PCR products in a third reaction using the outside primers, 1216-52 and 1842-97. [0234]
  • The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0235] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3728.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 5 and 6) of the fusion protein are shown in FIG. 7. [0236]
  • Fc-TMP-TMP. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a dimer of the TPO-mimetic peptide was constructed using standard PCR technology. Templates for PCR reactions were the pFc-A3 vector and a synthetic TMP-TMP gene. The synthetic gene was constructed from the 4 overlapping oligonucleotides (SEQ ID NOS: 371 to 374, respectively) shown below: [0237]
    1830-52 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCG
    ACT CTG CGT CAG TGG CTG GCT GCT CGT GCT
    1830-53 ACC TCC ACC ACC AGC ACG AGC AGC CAG CCA
    CTG ACG CAG AGT CGG ACC
    1830-54 GGT GGT GGA GGT GGC GGC GGA GGT ATT GAG
    GGC CCA ACC CTT CGC CAA TGG CTT GCA GCA
    CGC GCA
    1830-55 AAA AAA AGG ATC CTC GAG ATT ATG CGC GTG
    CTG CAA GCC ATT GGC GAA GGG TTG GGC CCT
    CAA TAC CTC CGC CGC C
  • The 4 oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 375 and 376, respectively) shown below: [0238]
    AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT
    1 ---------+---------+---------+---------+---------+---------+ 60
                            CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA
    a K  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A  R  A
    GGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCA
    61 ---------+---------+---------+---------+---------+---------+ 120
    CCACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGT
    a G  G  G  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A
    CGCGCA
    121 ---------------------------148
    GCGCGTATTAGAGCTCCTAGGAAAAAAA
    a R  A   *−
  • This duplex was amplified in a PCR reaction using 1830-52 and 1830-55 as the sense and antisense primers. [0239]
  • The Fc portion of the molecule was generated in a PCR reaction with pFc-A3 using the primers 1216-52 and 1830-51 as described above for Fc-TMP. The full length fusion gene was obtained from a third PCR reaction using the outside primers 1216-52 and 1830-55. [0240]
  • The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0241] E. coli strain 2596 cells as described in example 1. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3727.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 7 and 8) of the fusion protein are shown in FIG. 8. [0242]
  • TMP-TMP-Fc. A DNA sequence coding for a tandem repeat of the TPO-mimetic peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. Templates for PCR reactions were the EMP-Fc plasmid from strain #3688 (see Example 3) and a synthetic gene encoding the TMP dimer. The synthetic gene for the tandem repeat was constructed from the 7 overlapping oligonucleotides shown below (SEQ ID NOS: 377 to 383, respectively): [0243]
    1885-52
    TTT TTT CAT ATG ATC GAA GGT CCG ACT CTG CGT CAG
    TGG
    1885-53
    AGC ACG AGC AGC CAG CCA CTG ACG CAG AGT CGG ACC
    TTC GAT CAT ATG
    1885-54
    CTG GCT GCT CGT GCT GGT GGA GGC GGT GGG GAC AAA
    ACT CAC ACA
    1885-55
    CTG GCT GCT CGT GCT GGC GGT GGT GGC GGA GGG GGT
    GGC ATT GAG GGC CCA
    1885-56
    AAG CCA TTG GCG AAG GGT TGG GCC CTC AAT GCC ACC
    CCC TCC GCC ACC ACC GCC
    1885-57
    ACC CTT CGC CAA TGG CTT GCA GCA CGC GCA GGG GGA
    GGC GGT GGG GAC AAA ACT
    1885-58
    CCC ACC GCC TCC CCC TGC GCG TGC TGC
  • These oligonucleotides were annealed to form the duplex shown encoding an amino acid sequence shown below (SEQ ID NOS 384 and 385): [0244]
    TTTTTTCATATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGT
    1 ---------+---------+---------+---------+---------+---------+ 60
          GTATACTAGCTTCCAGGCTGAGACGCAGTCACCGACCGACGAGCACGACCGCCA
    a           M  I  E  G  P  T  L  R  Q  W  L  A  A  R  A  G  G
    GGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTGGCTGCTCGTGCT
    61 ---------+---------+---------+---------+---------+---------+ 120
    CCACCGCCTCCCCCACCGTAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGTGCGCGT
    a G  G  G  G  G  G  I  E  G  P  T  L  R  Q  W  L  A  A  R  A
    GGTGGAGGCGGTGGGGACAAAACTCTGGCTGCTCGTGCTGGTGGAGGCGGTGGGGACAAA
    121 ---------+---------+---------+---------+---------+---------+ 180
    CCCCCTCCGCCACCC
    a G  G  G  G  G  D  K  T  L  A  A  R  A  G  G  G  G  G  D  K
    ACTCACACA
    181 ---------  189
    a T  H  T    −
  • This duplex was amplified in a PCR reaction using 1885-52 and 1885-58 as the sense and antisense primers. [0245]
  • The Fc portion of the molecule was generated in a PCR reaction with DNA from the EMP-Fc fusion strain #3688 (see Example 3) using the primers 1885-54 and 1200-54. The full length fusion gene was obtained from a third PCR reaction using the outside primers 1885-52 and 1200-54. [0246]
  • The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0247] E. coli strain 2596 cells as described for Fc-EMP herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3798.
  • The nucelotide and amino acid sequences (SEQ ID NOS: 9 and 10) of the fusion protein are shown in FIG. 9. [0248]
  • TMP-Fc. A DNA sequence coding for a monomer of the TPO-mimetic peptide fused in-frame to the Fc region of human IgG1 was obtained fortuitously in the ligation in TMP-TMP-Fc, presumably due to the ability of primer 1885-54 to anneal to 1885-53 as well as to 1885-58. A single clone having the correct nucleotide sequence for the TMP-Fc construct was selected and designated Amgen strain #3788. [0249]
  • The nucleotide and amino acid sequences (SEQ ID NOS: 11 and 12) of the fusion protein are shown in FIG. 10. [0250]
  • Expression in [0251] E. coli. Cultures of each of the pAMG21-Fc-fusion constructs in E. coli GM221 were grown at 37° C. in Luria Broth medium containing 50 mg/ml kanamycin. Induction of gene product expression from the luxPR promoter was achieved following the addition of the synthetic autoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a final concentration of 20 ng/ml. Cultures were incubated at 37° C. for a further 3 hours. After 3 hours, the bacterial cultures were examined by microscopy for the presence of inclusion bodies and were then collected by centrifugation. Refractile inclusion bodies were observed in induced cultures indicating that the Fc-fusions were most likely produced in the insoluble fraction in E. coli. Cell pellets were lysed directly by resuspension in Laemmli sample buffer containing 10% b-mercaptoethanol and were analyzed by SDS-PAGE. In each case, an intense coomassie-stained band of the appropriate molecular weight was observed on an SDS-PAGE gel.
  • pAMG21. The expression plasmid pAMG21 can be derived from the Amgen expression vector pCFM1656 (ATCC #69576) which in turn be derived from the Amgen expression vector system described in U.S. Pat. No. 4,710,473. The pCFM1656 plasmid can be derived from the described pCFM836 plasmid (U.S. Pat. No. 4,710,473) by: [0252]
  • (a) destroying the two endogenous NdeI restriction sites by end filling with T4 polymerase enzyme followed by blunt end ligation; [0253]
  • (b) replacing the DNA sequence between the unique AatII and ClaI restriction sites containing the synthetic P[0254] L promoter with a similar fragment obtained from pCFM636 (U.S. Pat. No. 4,710,473) containing the PL promoter (see SEQ ID NO: 386 below); and
  • (c) substituting the small DNA sequence between the unique ClaI and KpnI restriction sites with the oligonucleotide having the sequence of SEQ ID NO: 388. [0255]
    AatII
    5′ CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT- SEQ ID NO: 386
    3′ TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA-
       -AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA-
       -TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT-
       -TACCACTGGCGGTGATACTGAGCACAT    3′:
       -ATGGTGACCGCCACTATGACTCGTGTAGC  5′
                                     ClaI
    5′ CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3′: SEQ ID NO: 387
    3′   TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC     5′
       ClaI                                               KpnI
  • The expression plasmid pAMG21 can then be derived from pCFM1656 by making a series of site-directed base changes by PCR overlapping oligo mutagenesis and DNA sequence substitutions. Starting with the BglII site (plasmid bp # 180) immediately 5′ to the plasmid replication promoter PcopB and proceeding toward the plasmid replication genes, the base pair changes are as shown in Table B below. [0256]
    TABLE B
    Base pair changes resulting in pAMG21
    pAMG21 bp # bp in pCFM1656 bp changed to in pAMG21
    # 204 T/A C/G
    # 428 A/T G/C
    # 509 G/C A/T
    # 617 insert two G/C bp
    # 679 G/C T/A
    # 980 T/A C/G
    # 994 G/C A/T
    # 1004 A/T C/G
    # 1007 C/G T/A
    # 1028 A/T T/A
    # 1047 C/G T/A
    # 1178 G/C T/A
    # 1466 G/C T/A
    # 2028 G/C bp deletion
    # 2187 C/G T/A
    # 2480 A/T T/A
    # 2499-2502 AGTG GTCA
    TCAC CAGT
    # 2642 TCCGAGC 7 bp deletion
    AGGCTCG
    # 3435 G/C A/T
    # 3446 G/C A/T
    # 3643 A/T T/A
  • The DNA sequence between the unique AatII (position #4364 in pCFM1656) and SacII (position #4585 in pCFM1656) restriction sites is substituted with the DNA sequence (SEQ ID NO: 23) shown in FIGS. 17A and 17B. During the ligation of the sticky ends of this substitution DNA sequence, the outside AatII and SacII sites are destroyed. There are unique AatII and SacII sites in the substituted DNA. [0257]
  • GM221 (Amgen #2596). The Amgen host strain #2596 is an [0258] E. coli K-12 strain derived from Amgen strain #393. It has been modified to contain both the temperature sensitive lambda repressor cI857s7 in the early ebg region and the lacIq repressor in the late ebg region (68 minutes). The presence of these two repressor genes allows the use of this host with a variety of expression systems, however both of these repressors are irrelevant to the expression from luxPR. The untransformed host has no antibiotic resistances.
  • The ribosome binding site of the cI857s7 gene has been modified to include an enhanced RBS. It has been inserted into the ebg operon between nucleotide position 1170 and 1411 as numbered in Genbank accession number M64441Gb_Ba with deletion of the intervening ebg sequence. The sequence of the insert is shown below with lower case letters representing the ebg sequences flanking the insert shown below [0259]
    (SEQ ID NO: 388)
    ttattttcgtGCGGCCGCACCATTATCACCGCCAGAGGTAAACTAGTCAACACGCACGGTGTTAGATATTTAT
    CCCTTGCGGTGATAGATTGAGCACATCGATTTGATTCTAGAAGGAGGGATAATATATGAGCACAAAAAAGAAA
    CCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTG
    GCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCAT
    CAATGCATTAAATGCTTATAACGCCGCATTGCTTACAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCT
    TCAATCGCCAGAGAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA
    CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTACCAAAGGTGATGCGGAG
    AGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCG
    CACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCC
    AGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAG
    GTGTTTTTACAACCACTAAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAG
    TTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGATAGACTAGTGGATCCACTAGTgtttctgccc:
  • The construct was delivered to the chromosome using a recombinant phage called MMebg-cI857s7enhanced RBS #4 into F′tet/393. After recombination and resolution only the chromosomal insert described above remains in the cell. It was renamed F′tet/GM101. F′tet/GM101 was then modified by the delivery of a lacI[0260] Q construct into the ebg operon between nucleotide position 2493 and 2937 as numbered in the Genbank accession number M64441Gb_Ba with the deletion of the intervening ebg sequence. The sequence of the insert is shown below with the lower case letters representing the ebg sequences flanking the insert (SEQ ID NO: 389) shown below:
    ggcggaaaccGACGTCCATCGAATGGTGCAAAACCTTTCGCGGTATGGCA
    TGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGT
    AACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTT
    CCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAA
    GTCGAAGCGGCGATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACA
    ACAACTGGCGGGCAAACAGTCGCTCCTGATTGGCGTTGCCACCTCCAGTC
    TGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCC
    GATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGCGT
    CGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTG
    GGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAA
    GCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGAC
    ACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCG
    TGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGC
    CCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATA
    TCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGA
    GTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATC
    GTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAAT
    GCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAG
    TGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACC
    ACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTT
    GCTGCAACTCTCTCAGGGCCAGCCGGTGAAGGGCAATCAGCTGTTGCCCG
    TCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCC
    TCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTC
    CCGACTGGAAAGCGGACAGTAAGGTACCATAGGATCCaggcacagga
  • The construct was delivered to the chromosome using a recombinant phage called AGebg-LacIQ#5 into F′tet/GM101. After recombination and resolution only the chromosomal insert described above remains in the cell. It was renamed F′tet/GM221. The F′tet episome was cured from the strain using acridine orange at a concentration of 25 μg/ml in LB. The cured strain was identified as tetracyline sensitive and was stored as GM221. [0261]
  • Expression. Cultures of pAMG21-Fc-TMP-TMP in [0262] E. coli GM221 in Luria Broth medium containing 50 μg/ml kanamycin were incubated at 37° C. prior to induction. Induction of Fc-TMP-TMP gene product expression from the luxPR promoter was achieved following the addition of the synthetic autoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a final concentration of 20 ng/ml and cultures were incubated at 37° C. for a further 3 hours. After 3 hours, the bacterial cultures were examined by microscopy for the presence of inclusion bodies and were then collected by centrifugation. Refractile inclusion bodies were observed in induced cultures indicating that the Fc-TMP-TMP was most likely produced in the insoluble fraction in E. coli. Cell pellets were lysed directly by resuspension in Laemmli sample buffer containing 10% •-mercaptoethanol and were analyzed by SDS-PAGE. An intense Coomassie stained band of approximately 30 kDa was observed on an SDS-PAGE gel. The expected gene product would be 269 amino acids in length and have an expected molecular weight of about 29.5 kDa. Fermentation was also carried out under standard batch conditions at the 10 L scale, resulting in similar expression levels of the Fc-TMP-TMP to those obtained at bench scale.
  • Purification of Fc-TMP-TMP. Cells are broken in water (1/10) by high pressure homogenization (2 passes at 14,000 PSI) and inclusion bodies are harvested by centrifugation (4200 RPM in J-6B for 1 hour). Inclusion bodies are solubilized in 6M guanidine, 50 mM Tris, 8 mM DTT, pH 8.7 for 1 hour at a 1/10 ratio. The solubilized mixture is diluted 20 times into 2M urea, 50 mM tris, 160 mM arginine, 3 mM cysteine, pH 8.5. The mixture is stirred overnight in the cold and then concentrated about 10 fold by ultafiltration. It is then diluted 3 fold with 10 mM Tris, 1.5M urea, pH 9. The pH of this mixture is then adjusted to pH 5 with acetic acid. The precipitate is removed by centrifugation and the supernatant is loaded onto a SP-Sepharose Fast Flow column equilibrated in 20 mM NaAc, 100 mM NaCl, pH 5(10 mg/ml protein load, room temperature). The protein is eluted off using a 20 column volume gradient in the same buffer ranging from 100 mM NaCl to 500 mM NaCl. The pool from the column is diluted 3 fold and loaded onto a SP-Sepharose HP column in 20 mM NaAc, 150 mM NaCl, pH 5(10 mg/ml protein load, room temperature). The protein is eluted off using a 20 column volume gradient in the same buffer ranging from 150 mM NaCl to 400 mM NaCl. The peak is pooled and filtered. [0263]
  • Characterization of Fc-TMP activity. The following is a summary of in vivo data in mice with various compounds of this invention. [0264]
  • Mice: Normal female BDF1 approximately 10-12 weeks of age. [0265]
  • Bleed schedule: Ten mice per group treated on day 0, two groups started 4 days apart for a total of 20 mice per group. Five mice bled at each time point, mice were bled a minimum of three times a week. Mice were anesthetized with isoflurane and a total volume of 140-160 μl of blood was obtained by puncture of the orbital sinus. Blood was counted on a Technicon H1E blood analyzer running software for murine blood. Parameters measured were white blood cells, red blood cells, hematocrit, hemoglobin, platelets, neutrophils. [0266]
  • Treatments: Mice were either injected subcutaneously for a bolus treatment or implanted with 7-day micro-osmotic pumps for continuous delivery. Subcutaneous injections were delivered in a volume of 0.2 ml. Osmotic pumps were inserted into a subcutaneous incision made in the skin between the scapulae of anesthetized mice. Compounds were diluted in PBS with 0.1% BSA. All experiments included one control group, labeled “carrier” that were treated with this diluent only. The concentration of the test articles in the pumps was adjusted so that the calibrated flow rate from the pumps gave the treatment levels indicated in the graphs. [0267]
  • Compounds: A dose titration of the compound was delivered to mice in 7 day micro-osmotic pumps. Mice were treated with various compounds at a single dose of 100 μg/kg in 7 day osmotic pumps. Some of the same compounds were then given to mice as a single bolus injection. [0268]
  • Activity test results: The results of the activity experiments are shown in FIGS. 11 and 12. In dose response assays using 7-day micro-osmotic pumps, the maximum effect was seen with the compound of SEQ ID NO: 18 was at 100 μg/kg/day; the 10 μg/kg/day dose was about 50% maximally active and 1 μg/kg/day was the lowest dose at which activity could be seen in this assay system. The compound at 10 μg/kg/day dose was about equally active as 100 μg/kg/day unpegylated rHu-MGDF in the same experiment. [0269]
  • Example 3 Fc-EMP Fusions
  • Fc-EMP. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of the EPO-mimetic peptide was constructed using standard PCR technology. Templates for PCR reactions were a vector containing the Fc sequence (pFc-A3, described in International application WO 97/23614, published Jul. 3, 1997) and a synthetic gene encoding EPO monomer. The synthetic gene for the monomer was constructed from the 4 overlapping oligonucleotides (SEQ ID NOS: 390 to 393, respectively) shown below: [0270]
    1798-2
    TAT GAA AGG TGG AGG TGG TGG TGG AGG TAC TTA CTC
    TTG CCA CTT CGG CCC GCT GAC TTG G
    1798-3
    CGG TTT GCA AAC CCA AGT CAG CGG GCC GAA GTG GCA
    AGA GTA AGT ACC TCC ACC ACC ACC TCC ACC TTT CAT
    1798-4
    GTT TGC AAA CCG CAG GGT GGC GGC GGC GGC GGC GGT
    GGT ACC TAT TCC TGT CAT TTT
    1798-5
    CCA GGT CAG CGG GCC AAA ATG ACA GGA ATA GGT ACC
    ACC GCC GCC GCC GCC GCC ACC CTG
  • The 4 oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 394 and 395, respectively) shown below: [0271]
    TATGAAAGGTGGAGGTGGTGGTGGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTG
    1 ---------+---------+---------+---------+---------+---------+ 60
    TACTTTCCACCTCCACCACCACCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAAC
    b  M  K  G  G  G  G  G  G  G  T  Y  S  C  H  F  G  P  L  T  W
    GGTTTGCAAACCGCAGGGTGGCGGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTT
    61 ---------+---------+---------+---------+---------+----------+----------+-- 133
    CCAAACGTTTGGCGTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTGGACC
    b  V  C  K  P  Q  G  G  G  G  G  G  G  G  T  Y  S  C  H  F
  • This duplex was amplified in a PCR reaction using [0272]
    1798-18
    GCA GAA GAG CCT CTC CCT GTC TCC GGG TAA AGG TGG
    AGG TGG TGG TGG AGG TAC TTA CTC T and
    1798-19
    CTA ATT GGA TCC ACG AGA TTA ACC ACC CTG CGG TTT
    GCA A
  • as the sense and antisense primers (SEQ ID NOS: 396 and 397, respectively). [0273]
  • The Fc portion of the molecule was generated in a PCR reaction with pFc-A3 using the primers [0274]
    1216-52
    AAC ATA AGT ACC TGT AGG ATC G
    1798-17
    AGA GTA AGT ACC TCC ACC ACC ACC TCC ACC TTT ACC
    CGG AGA CAG GGA GAG GCT CTT CTG C
  • which are SEQ ID NOS: 398 and 399, respectively. The oligonucleotides 1798-17 and 1798-18 contain an overlap of 61 nucleotides, allowing the two genes to be fused together in the correct reading frame by combining the above PCR products in a third reaction using the outside primers, 1216-52 and 1798-19. [0275]
  • The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 (described below), also digested with XbaI and BamHI. Ligated DNA was transformed into competent host cells of [0276] E. coli strain 2596 (GM221, described herein). Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3718.
  • The nucleotide and amino acid sequence of the resulting fusion protein (SEQ ID NOS: 15 and 16) are shown in FIG. 13. [0277]
  • EMP-Fc. A DNA sequence coding for a monomer of the EPO-mimetic peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. Templates for PCR reactions were the pFC-A3a vector and a synthetic gene encoding EPO monomer. The synthetic gene for the monomer was constructed from the 4 overlapping oligonucleotides 1798-4 and 1798-5 (above) and 1798-6 and 1798-7 (SEQ ID NOS: 400 and 401, respectively) shown below: [0278]
    1798-6
    GGC CCG CTG ACC TGG GTA TGT AAG CCA CAA GGG GGT
    GGG GGA GGC GGG GGG TAA TCT CGA G
    1798-7
    GAT CCT CGA GAT TAC CCC CCG CCT CCC CCA CCC CCT
    TGT GGC TTA CAT AC
  • The 4 oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 402 and 403, respectively) shown below: [0279]
    GTTTGCAAACCGCAGGGTGGCGGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGC
    1 ---------+---------+---------+---------+---------+---------+ 60
                GTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCG
    A V  C  K  P  Q  G  G  G  G  G  G  G  G  T  Y  S  C  H  F  G
    CCGCTGACCTGGGTATGTAAGCCACAAGGGGGTGGGGGAGGCGGGGGGTAATCTCGAG
    61 ---------+---------+---------+---------+---------+---------+- 122
    GGCGACTGGACCCATACATTCGGTGTTCCCCCACCCCCTCCGCCCCCCATTAGAGCTCCTAG
    A P  L  T  W  V  C  K  P  Q  G  G  G  G  G  G  G  *
  • This duplex was amplified in a PCR reaction using [0280]
    1798-21
    TTA TTT CAT ATG AAA GGT GGT AAC TAT TCC TGT CAT
    TTT and
    1798-22
    TGG ACA TGT GTG AGT TTT GTC CCC CCC GCC TCC CCC
    ACC CCC T
  • as the sense and antisense primers (SEQ ID NOS: 404 and 405, respectively). [0281]
  • The Fc portion of the molecule was generated in a PCR reaction with pFc-A3 using the primers [0282]
    1798-23
    AGG GGG TGG GGG AGG CGG GGG GGA CAA AAC TCA CAC
    ATG TCC A and
    1200-54
    GTT ATT GCT CAG CGG TGG CA
  • which are SEQ ID NOS: 406 and 407, respectively. The oligonucleotides 1798-22 and 1798-23 contain an overlap of 43 nucleotides allowing the two genes to be fused together in the correct reading frame by combining the above PCR products in a third reaction using the outside primers, 1787-21 and 1200-54. [0283]
  • The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0284] E. coli strain 2596 cells as described above. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3688.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 17 and 18) of the resulting fusion protein are shown in FIG. 14. [0285]
  • EMP-EMP-Fc. A DNA sequence coding for a dimer of the EPO-mimetic peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. Templates for PCR reactions were the EMP-Fc plasmid from strain #3688 above and a synthetic gene encoding the EPO dimer. The synthetic gene for the dimer was constructed from the 8 overlapping oligonucleotides (SEQ ID NOS:408 to 415, respectively) shown below: [0286]
    1869-23
    TTT TTT ATC GAT TTG ATT CTA GAT TTG AGT TTT AAC
    TTT TAG AAG GAG GAA TAA AAT ATG
    1869-48
    TAA AAG TTA AAA CTC AAA TCT AGA ATC AAA TCG ATA
    AAA AA
    1871-72
    GGA GGT ACT TAC TCT TGC CAC TTC GGC CCG CTG ACT
    TGG GTT TGC AAA CCG
    1871-73
    AGT CAG CGG GCC GAA GTG GCA AGA GTA AGT ACC TCC
    CAT ATT TTA TTC CTC CTT C
    1871-74
    CAG GGT GGC GGC GGC GGC GGC GGT GGT ACC TAT TCC
    TGT CAT TTT GGC CCG CTG ACC TGG
    1871-75
    AAA ATG ACA GGA ATA GGT ACC ACC GCC GCC GCC GCC
    GCC ACC CTG CGG TTT GCA AAC CCA
    1871-78
    GTA TGT AAG CCA CAA GGG GGT GGG GGA GGC GGG GGG
    GAC AAA ACT CAC ACA TGT CCA
    1871-79
    AGT TTT GTC CCC CCC GCC TCC CCC ACC CCC TTG TGG
    CTT ACA TAC CCA GGT CAG CGG GCC
  • The 8 oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 416 and 417, respectively) shown below: [0287]
    TTTTTTATCGATTTGATTCTAGATTTGAGTTTTAACTTTTAGAAGGAGGAATAAAATATG
    1 ---------+---------+---------+---------+---------+---------+ 60
    AAAAAATAGCTAAACTAAGATCTAAACTCAAAATTGAAAATCTTCCTCCTTATTTTATAC
    a                                                           M-
    GGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTGGGTTTGCAAACCGCAGGGTGGC
    61 ---------+---------+---------+---------+---------+---------+ 120
    CCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAACCCAAACGTTTGGCGTCCCACCG
    a G  G  T  Y  S  C  H  F  G  P  L  T  W  V  C  K  P  Q  G  G
    GGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGCCCGCTGACCTGGGTATGTAAG
    121 ---------+---------+---------+---------+---------+---------+ 180
    CCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTGGACCCATACATTC
    a G  G  G  G  G  G  T  Y  S  C  H  F  G  P  L  T  W  V  C  K
    CCACAAGGGGGTGGGGGAGGCGGGGGGGACAAAACTCACACATGTCCA
    181 ---------+---------+---------+---------+--------  228
    GGTGTTCCCCCACCCCCTCCGCCCCCCCTGTTTTGA
    a P  Q  G  G  G  G  G  G  G  D  K  T  H  T  C  P    −
  • This duplex was amplified in a PCR reaction using 1869-23 and 1871-79 (shown above) as the sense and antisense primers. [0288]
  • The Fc portion of the molecule was generated in a PCR reaction with strain 3688 DNA using the primers 1798-23 and 1200-54 (shown above). [0289]
  • The oligonucleotides 1871-79 and 1798-23 contain an overlap of 31 nucleotides, allowing the two genes to be fused together in the correct reading frame by combining the above PCR products in a third reaction using the outside primers, 1869-23 and 1200-54. [0290]
  • The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0291] E. coli strain 2596 cells as described for Fc-EMP. Clones were screened for ability to produce the recombinant protein product and possession of the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3813.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 19 and 20, respectively) of the resulting fusion protein are shown in FIG. 15. There is a silent mutation at position 145 (A to G, shown in boldface) such that the final construct has a different nucleotide sequence than the oligonucleotide 1871-72 from which it was derived. [0292]
  • Fc-EMP-EMP. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a dimer of the EPO-mimetic peptide was constructed using standard PCR technology. Templates for PCR reactions were the plasmids from strains 3688 and 3813 above. [0293]
  • The Fc portion of the molecule was generated in a PCR reaction with strain 3688 DNA using the primers 1216-52 and 1798-17 (shown above). The EMP dimer portion of the molecule was the product of a second PCR reaction with strain 3813 DNA using the primers 1798-18 (also shown above) and SEQ ID NO: 418, shown below: [0294]
  • 1798-20 CTA ATT GGA TCC TCG AGA TTA ACC CCC TTG TGG CTT ACAT [0295]
  • The oligonucleotides 1798-17 and 1798-18 contain an overlap of 61 nucleotides, allowing the two genes to be fused together in the correct reading frame by combining the above PCR products in a third reaction using the outside primers, 1216-52 and 1798-20. [0296]
  • The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0297] E. coli strain 2596 cells as described for Fc-EMP. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3822.
  • The nucleotide and amino acid sequences (SEQ ID NOS: ______ and ______, respectively) of the fusion protein are shown in FIG. 16. [0298]
  • Characterization of Fc-EMP activity. Characterization was carried out in vivo as follows. [0299]
  • Mice: Normal female BDF1 approximately 10-12 weeks of age. [0300]
  • Bleed schedule: Ten mice per group treated on day 0, two groups started 4 days apart for a total of 20 mice per group. Five mice bled at each time point, mice were bled a maximum of three times a week. Mice were anesthetized with isoflurane and a total volume of 140-160 ml of blood was obtained by puncture of the orbital sinus. Blood was counted on a Technicon H1E blood analyzer running software for murine blood. Parameters measured were WBC, RBC, HCT, HGB, PLT, NEUT, LYMPH. [0301]
  • Treatments: Mice were either injected subcutaneously for a bolus treatment or implanted with 7 day micro-osmotic pumps for continuous delivery. Subcutaneous injections were delivered in a volume of 0.2 ml. Osmotic pumps were inserted into a subcutaneous incision made in the skin between the scapulae of anesthetized mice. Compounds were diluted in PBS with 0.1% BSA. All experiments included one control group, labeled “carrier” that were treated with this diluent only. The concentration of the test articles in the pumps was adjusted so that the calibrated flow rate from the pumps gave the treatment levels indicated in the graphs. [0302]
  • Experiments: Various Fc-conjugated EPO mimetic peptides (EMPs) were delivered to mice as a single bolus injection at a dose of 100 μg/kg. Fc-EMPs were delivered to mice in 7-day micro-osmotic pumps. The pumps were not replaced at the end of 7 days. Mice were bled until day 51 when HGB and HCT returned to baseline levels. [0303]
  • Example 4 TNF-α Inhibitors
  • Fc-TNF-α inhibitors. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of the TNF-α inhibitory peptide was constructed using standard PCR technology. The Fc and 5 glycine linker portion of the molecule was generated in a PCR reaction with DNA from the Fc-EMP fusion strain #3718 (see Example 3) using the sense primer 1216-52 and the antisense primer 2295-89 (SEQ ID NOS: 1112 and 1113, respectively). The nucleotides encoding the TNF-α inhibitory peptide were provided by the PCR primer 2295-89 shown below: [0304]
    1216-52
    AAC ATA AGT ACC TGT AGG ATC G
    2295-89
    CCG CGG ATC CAT TAC GGA CGG TGA CCC AGA GAG GTG
    TTT TTG TAG TGC GGC AGG AAG TCA CCA CCA CCT CCA
    CCT TTA CCC
  • The oligonucleotide 2295-89 overlaps the glycine linker and Fc portion of the template by 22 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame. [0305]
  • The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0306] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4544.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 1055 and 1056) of the fusion protein are shown in FIGS. 19A and 19B. [0307]
  • TNF-α inhibitor-Fc. A DNA sequence coding for a TNF-α inhibitory peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. The template for the PCR reaction was a plasmid containing an unrelated peptide fused via a five glycine linker to Fc. The nucleotides encoding the TNF-α inhibitory peptide were provided by the sense PCR primer 2295-88, with primer 1200-54 serving as the antisense primer (SEQ ID NOS: 1117 and 407, respectively). The primer sequences are shown below: [0308]
    2295-88
    GAA TAA CAT ATG GAC TTC CTG CCG CAC TAC AAA AAC
    ACC TCT CTG GGT CAC CGT CCG GGT GGA GGC GGT GGG
    GAC AAA ACT
    1200-54
    GTT ATT GCT CAG CGG TGG CA
  • The oligonucleotide 2295-88 overlaps the glycine linker and Fc portion of the template by 24 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame. [0309]
  • The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0310] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4543.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 1057 and 1058) of the fusion protein are shown in FIGS. 20A and 20B. [0311]
  • Expression in [0312] E. coli. Cultures of each of the pAMG21-Fc-fusion constructs in E. coli GM221 were grown at 37° C. in Luria Broth medium containing 50 mg/ml kanamycin. Induction of gene product expression from the luxPR promoter was achieved following the addition of the synthetic autoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a final concentration of 20 ng/ml. Cultures were incubated at 37° C. for a further 3 hours. After 3 hours, the bacterial cultures were examined by microscopy for the presence of inclusion bodies and were then collected by centrifugation. Refractile inclusion bodies were observed in induced cultures indicating that the Fc-fusions were most likely produced in the insoluble fraction in E. coli. Cell pellets were lysed directly by resuspension in Laemmli sample buffer containing 10% β-mercaptoethanol and were analyzed by SDS-PAGE. In each case, an intense coomassie-stained band of the appropriate molecular weight was observed on an SDS-PAGE gel.
  • Purification of Fc-peptide fusion proteins. Cells are broken in water ({fraction (1/10)}) by high pressure homogenization (2 passes at 14,000 PSI) and inclusion bodies are harvested by centrifugation (4200 RPM in J-6B for 1 hour). Inclusion bodies are solubilized in 6M guanidine, 50 mM Tris, 8 mM DTT, pH 8.7 for 1 hour at a {fraction (1/10)} ratio. The solubilized mixture is diluted 20 times into 2M urea, 50 mM tris, 160 mM arginine, 3 mM cysteine, pH 8.5. The mixture is stirred overnight in the cold and then concentrated about 10 fold by ultafiltration. It is then diluted 3 fold with 10 mM Tris, 1.5M urea, pH 9. The pH of this mixture is then adjusted to pH 5 with acetic acid. The precipitate is removed by centrifugation and the supernatant is loaded onto a SP-Sepharose Fast Flow column equilibrated in 20 mM NaAc, 100 mM NaCl, pH 5 (10 mg/ml protein load, room temperature). The protein is eluted from the column using a 20 column volume gradient in the same buffer ranging from 100 mM NaCl to 500 mM NaCl. The pool from the column is diluted 3 fold and loaded onto a SP-Sepharose HP column in 20 mM NaAc, 150 mM NaCl, pH 5(10 mg/ml protein load, room temperature). The protein is eluted using a 20 column volume gradient in the same buffer ranging from 150 mM NaCl to 400 mM NaCl. The peak is pooled and filtered. [0313]
  • Characterization of activity of Fc-TNF-α inhibitor and TNF-α inhibitor-Fc. Binding of these peptide fusion proteins to TNF-(x can be characterized by BIAcore by methods available to one of ordinary skill in the art who is armed with the teachings of the present specification. [0314]
  • Example 5 IL-1 Antagonists
  • Fc-IL-1 antagonist. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of an IL-1 antagonist peptide was constructed using standard PCR technology. The Fc and 5 glycine linker portion of the molecule was generated in a PCR reaction with DNA from the Fc-EMP fusion strain #3718 (see Example 3) using the sense primer 1216-52 and the antisense primer 2269-70 (SEQ ID NOS: 1112 and 1118, respectively). The nucleotides encoding the IL-1 antagonist peptide were provided by the PCR primer 2269-70 shown below: [0315]
    1216-52
    AAC ATA AGT ACC TGT AGG ATC G
    2269-70
    CCG CGG ATC CAT TAC AGC GGC AGA GCG TAC GGC TGC
    CAG TAA CCC GGG GTC CAT TCG AAA CCA CCA CCT CCA
    CCT TTA CCC
  • The oligonucleotide 2269-70 overlaps the glycine linker and Fc portion of the template by 22 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame. [0316]
  • The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0317] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4506.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 1059 and 1060) of the fusion protein are shown in FIGS. 21A and 21B. [0318]
  • IL-1 antagonist-Fc. A DNA sequence coding for an IL-1 antagonist peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. The template for the PCR reaction was a plasmid containing an unrelated peptide fused via a five glycine linker to Fc. The nucleotides encoding the IL-1 antagonist peptide were provided by the sense PCR primer 2269-69, with primer 1200-54 serving as the antisense primer (SEQ ID NOS: 1119 and 407, respectively). The primer sequences are shown below: [0319]
    2269-69
    GAA TAA CAT ATG TTC GAA TGG ACC CCG GGT TAC TGG
    CAG CCG TAC GCT CTG CCG CTG GGT GGA GGC GGT GGG
    GAC AAA ACT
    1200-54
    GTT ATT GCT CAG CGG TGG CA
  • The oligonucleotide 2269-69 overlaps the glycine linker and Fc portion of the template by 24 nucleotides, with the PCR resulting in the two genes 35 being fused together in the correct reading frame. [0320]
  • The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0321] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4505.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 1061 and 1062) of the fusion protein are shown in FIGS. 22A and 22B. Expression and purification were carried out as in previous examples. [0322]
  • Characterization of Fc-IL-1 antagonist peptide and IL-1 antagonist peptide-Fc activity. IL-1 Receptor Binding competition between IL-1β, IL-1RA and Fc-conjugated IL-1 peptide sequences was carried out using the IGEN system. Reactions contained 0.4 nM biotin-IL-1R+15 nM IL-1-TAG+3 uM competitor+20 ug/ml streptavidin-conjugate beads, where competitors were IL-1RA, Fc-IL-1 antagonist, IL-1 antagonist-Fc). Competition was assayed over a range of competitor concentrations from 3 uM to 1.5 pM. The results are shown in Table C below: [0323]
    TABLE C
    Results from IL-1 Receptor Binding Competition Assay
    IL-1pep-Fc Fc-IL-1pep IL-1ra
    KI 281.5 59.58 1.405
    EC50 530.0 112.2 2.645
    95% Confidence Intervals
    EC50 280.2 to 1002 54.75 to 229.8 1.149 to
    6.086
    KI 148.9 to 532.5 29.08 to 122.1 0.6106 to
    3.233
    Goodness of Fit
    R2 0.9790 0.9687 0.9602
  • Example 6 VEGF-Antagonists
  • Fc-VEGF Antagonist. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of the VEGF mimetic peptide was constructed using standard PCR technology. The templates for the PCR reaction were the pFc-A3 plasmid and a synthetic VEGF mimetic peptide gene. The synthetic gene was assembled by annealing the following two oligonucleotides primer (SEQ ID NOS: 1120 and 1121, respectively): [0324]
    2293-11
    GTT GAA CCG AAC TGT GAC ATC CAT GTT ATG TGG GAA
    TGG GAA TGT TTT GAA CGT CTG
    2293-12
    CAG ACG TTC AAA ACA TTC CCA TTC CCA CAT AAC ATG
    GAT GTC ACA GTT CGG TTC AAC
  • The two oligonucleotides anneal to form the following duplex encoding an amino acid sequence shown below (SEQ ID NOS 1122): [0325]
    GTTGAACCGAACTGTGACATCCATGTTATGTGGGAATGGGAATGTTTTGAACGTCTG
    1 ---------+---------+---------+---------+---------+------- 57
    CAACTTGGCTTGACACTGTAGGTACAATACACCCTTACCCTTACAAAACTTGCAGAC
    a V  E  P  N  C  D  I  H  V  M  W  E  W  E  C  F  E  R  L
  • This duplex was amplified in a PCR reaction using 2293-05 and 2293-06 as the sense and antisense primers (SEQ ID NOS. 1125 and 1126). [0326]
  • The Fc portion of the molecule was generated in a PCR reaction with the pFc-A3 plasmid using the primers 2293-03 and 2293-04 as the sense and antisense primers (SEQ ID NOS. 1123 and 1124, respectively). The full length fusion gene was obtained from a third PCR reaction using the outside primers 2293-03 and 2293-06. These primers are shown below: [0327]
    2293-03 ATT TGA TTC TAG AAG GAG GAA TAA CAT ATG
    GAC AAA ACT CAC ACA TGT
    2293-04 GTC ACA GTT CGG TTC AAC ACC ACC ACC ACC
    ACC TTT ACC CGG AGA CAG GGA
    2293-05 TCC CTG TCT CCG GGT AAA GGT GGT GGT GGT
    GGT GTT GAA CCG AAC TGT GAC ATC
    2293-06 CCG CGG ATC CTC GAG TTA CAG ACG TTC AAA
    ACA TTC CCA
  • The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0328] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4523.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 1063 and 1064) of the fusion protein are shown in FIGS. 23A and 23B. [0329]
  • VEGF antagonist-Fc. A DNA sequence coding for a VEGF mimetic peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. The templates for the PCR reaction were the pFc-A3 plasmid and the synthetic VEGF mimetic peptide gene described above. The synthetic duplex was amplified in a PCR reaction using 2293-07 and 2293-08 as the sense and antisense primers (SEQ ID NOS. 1127 and 1128, respectively). [0330]
  • The Fc portion of the molecule was generated in a PCR reaction with the pFc-A3 plasmid using the primers 2293-09 and 2293-10 as the sense and antisense primers (SEQ ID NOS. 1129 and 1130, respectively). The full length fusion gene was obtained from a third PCR reaction using the outside primers 2293-07 and 2293-10. These primers are shown below: [0331]
    2293-07 ATT TGA TTC TAG AAG GAG GAA TAA CAT ATG
    GTT GAA CCG AAC TGT GAC
    2293-08 ACA TGT GTG AGT TTT GTC ACC ACC ACC ACC
    ACC CAG ACG TTC AAA ACA TTC
    2293-09 GAA TGT TTT GAA CGT CTG GGT GGT GGT GGT
    GGT GAC AAA ACT CAC ACA TGT
    2293-10 CCG CGG ATC CTC GAG TTA TTT ACC CGG AGA
    CAG GGA GAG
  • The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0332] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4524.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 1065 and 1066) of the fusion protein are shown in FIGS. 24A and 24B. Expression and purification were carried out as in previous examples. [0333]
  • Example 7 MMP Inhibitors
  • Fc-MMP inhibitor. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of an MMP inhibitory peptide was constructed using standard PCR technology. The Fc and 5 glycine linker portion of the molecule was generated in a PCR reaction with DNA from the Fc-TNF-α inhibitor fusion strain #4544 (see Example 4) using the sense primer 1216-52 and the antisense primer 2308-67 (SEQ ID NOS: 1112 and 1131, respectively). The nucleotides encoding the MMP inhibitor peptide were provided by the PCR primer 2308-67 shown below: [0334]
    1216-52 AAC ATA AGT ACC TGT AGG ATC G
    2308-67 CCG CGG ATC CAT TAG CAC AGG GTG AAA CCC
    CAG TGG GTG GTG CAA CCA CCA CCT CCA CCT
    TTA CCC
  • The oligonucleotide 2308-67 overlaps the glycine linker and Fc portion of the template by 22 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame. [0335]
  • The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0336] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4597.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 1067 and 1068) of the fusion protein are shown in FIGS. 25A and 25B. Expression and purification were carried out as in previous examples. [0337]
  • MMP Inhibitor-Fc. A DNA sequence coding for an MMP inhibitory peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. The Fc and 5 glycine linker portion of the molecule was generated in a PCR reaction with DNA from the Fc-TNF-α inhibitor fusion strain #4543 (see Example 4). The nucleotides encoding the MMP inhibitory peptide were provided by the sense PCR primer 2308-66, with primer 1200-54 serving as the antisense primer (SEQ ID NOS: 1132 and 407, respectively). The primer sequences are shown below: [0338]
    2308-66 GAA TAA CAT ATG TGC ACC ACC CAC TGG GGT
    TTC ACC CTG TGC GGT GGA GGC GGT GGG GAC
    AAA
    1200-54 GTT ATT GCT CAG CGG TGG CA
  • The oligonucleotide 2269-69 overlaps the glycine linker and Fc portion of the template by 24 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame. [0339]
  • The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent [0340] E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4598.
  • The nucleotide and amino acid sequences (SEQ ID NOS: 1069 and 1070) of the fusion protein are shown in FIGS. 26A and 26B. [0341]
  • The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto, without departing from the spirit and scope of the invention as set forth herein. [0342]
  • Abbreviations
  • Abbreviations used throughout this specification are as defined below, unless otherwise defined in specific circumstances. [0343]
    Ac acetyl (used to refer to acetylated residues)
    AcBpa acetylated p-benzoyl-L-phenylalanine
    ADCC antibody-dependent cellular cytotoxicity
    Aib aminoisobutyric acid
    bA beta-alanine
    Bpa p-benzoyl-L-phenylalanine
    BrAc bromoacetyl (BrCH2C(O)
    BSA Bovine serum albumin
    Bzl Benzyl
    Cap Caproic acid
    CTL Cytotoxic T lymphocytes
    CTLA4 Cytotoxic T lymphocyte antigen 4
    DARC Duffy blood group antigen receptor
    DCC Dicylcohexylcarbodiimide
    Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl
    EMP Erythropoietin-mimetic peptide
    ESI-MS Electron spray ionization mass spectrometry
    EPO Erythropoietin
    Fmoc fluorenylmethoxycarbonyl
    G-CSF Granulocyte colony stimulating factor
    GH Growth hormone
    HCT hematocrit
    HGB hemoglobin
    hGH Human growth hormone
    HOBt 1-Hydroxybenzotriazole
    HPLC high performance liquid chromatography
    IL interleukin
    IL-R interleukin receptor
    IL-1R interleukin-1 receptor
    IL-1ra interleukin-1 receptor antagonist
    Lau Lauric acid
    LPS lipopolysaccharide
    LYMPH lymphocytes
    MALDI-MS Matrix-assisted laser desorption ionization mass
    spectrometry
    Me methyl
    MeO methoxy
    MHC major histocompatibility complex
    MMP matrix metalloproteinase
    MMPI matrix metalloproteinase inhibitor
    1-Nap 1-napthylalanine
    NEUT neutrophils
    NGF nerve growth factor
    Nle norleucine
    NMP N-methyl-2-pyrrolidinone
    PAGE polyacrylamide gel electrophoresis
    PBS Phosphate-buffered saline
    Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl
    PCR polymerase chain reaction
    Pec pipecolic acid
    PEG Poly(ethylene glycol)
    pGlu pyroglutamic acid
    Pic picolinic acid
    PLT platelets
    pY phosphotyrosine
    RBC red blood cells
    RBS ribosome binding site
    RT room temperature (25° C.)
    Sar sarcosine
    SDS sodium dodecyl sulfate
    STK serine-threonine kinases
    t-Boc tert-Butoxycarbonyl
    tBu tert-Butyl
    TGF tissue growth factor
    THF thymic humoral factor
    TK tyrosine kinase
    TMP Thrombopoietin-mimetic peptide
    TNF Tissue necrosis factor
    TPO Thrombopoietin
    TRAIL TNF-related apoptosis-inducing ligand
    Trt trityl
    UK urokinase
    UKR urokinase receptor
    VEGF vascular endothelial cell growth factor
    VIP vasoactive intestinal peptide
    WBC white blood cells
  • [0344]
  • 0
    SEQUENCE LISTING
    <160> NUMBER OF SEQ ID NOS: 1133
    <210> SEQ ID NO 1
    <211> LENGTH: 684
    <212> TYPE: DNA
    <213> ORGANISM: HUMAN
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (1)..(684)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 1
    atg gac aaa act cac aca tgt cca cct tgt cca gct ccg gaa ctc ctg 48
    Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
    1 5 10 15
    ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 96
    Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
    20 25 30
    atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 144
    Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
    35 40 45
    cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 192
    His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
    50 55 60
    gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg 240
    Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
    65 70 75 80
    tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat 288
    Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
    85 90 95
    ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc 336
    Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
    100 105 110
    atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag 384
    Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
    115 120 125
    gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc 432
    Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
    130 135 140
    agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 480
    Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
    145 150 155 160
    gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 528
    Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
    165 170 175
    ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 576
    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
    180 185 190
    gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg 624
    Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
    195 200 205
    atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg 672
    Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
    210 215 220
    tct ccg ggt aaa 684
    Ser Pro Gly Lys
    225
    <210> SEQ ID NO 2
    <211> LENGTH: 228
    <212> TYPE: PRT
    <213> ORGANISM: HUMAN
    <400> SEQUENCE: 2
    Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
    1 5 10 15
    Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
    20 25 30
    Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
    35 40 45
    His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
    50 55 60
    Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
    65 70 75 80
    Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
    85 90 95
    Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
    100 105 110
    Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
    115 120 125
    Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
    130 135 140
    Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
    145 150 155 160
    Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
    165 170 175
    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
    180 185 190
    Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
    195 200 205
    Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
    210 215 220
    Ser Pro Gly Lys
    225
    <210> SEQ ID NO 3
    <211> LENGTH: 36
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC SCHEME FOR PREPARATION OF PEGYLATED
    PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (18)..(18)
    <223> OTHER INFORMATION: Methoxy-polyethylene glycol (5000
    Dalton)-sulfoacetyl group attached to the sidechain.
    <400> SEQUENCE: 3
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly
    1 5 10 15
    Gly Lys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu
    20 25 30
    Ala Ala Arg Ala
    35
    <210> SEQ ID NO 4
    <211> LENGTH: 36
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: SYNTHETIC SCHEME FOR PREPARATION OF PEGYLATED
    PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (18)..(18)
    <223> OTHER INFORMATION: Methoxy-polyethylene glycol (5000
    Dalton)-succinimidyl group attached to the sidechain.
    <400> SEQUENCE: 4
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly
    1 5 10 15
    Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu
    20 25 30
    Ala Ala Arg Ala
    35
    <210> SEQ ID NO 5
    <211> LENGTH: 794
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Fc-TMP
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (39)..(779)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 5
    tctagatttg ttttaactaa ttaaaggagg aataacat atg gac aaa act cac aca 56
    Met Asp Lys Thr His Thr
    1 5
    tgt cca cct tgt cca gct ccg gaa ctc ctg ggg gga ccg tca gtc ttc 104
    Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
    10 15 20
    ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 152
    Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
    25 30 35
    gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 200
    Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
    40 45 50
    aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 248
    Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
    55 60 65 70
    aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc 296
    Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
    75 80 85
    ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc 344
    Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
    90 95 100
    aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc 392
    Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
    105 110 115
    aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca 440
    Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
    120 125 130
    tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc 488
    Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
    135 140 145 150
    aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 536
    Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
    155 160 165
    cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 584
    Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
    170 175 180
    ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg 632
    Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
    185 190 195
    cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac 680
    Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
    200 205 210
    aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa ggt gga 728
    Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly
    215 220 225 230
    ggt ggt ggt atc gaa ggt ccg act ctg cgt cag tgg ctg gct gct cgt 776
    Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg
    235 240 245
    gct taatctcgag gatcc 794
    Ala
    <210> SEQ ID NO 6
    <211> LENGTH: 247
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Fc-TMP
    <400> SEQUENCE: 6
    Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
    1 5 10 15
    Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
    20 25 30
    Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
    35 40 45
    His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
    50 55 60
    Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
    65 70 75 80
    Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
    85 90 95
    Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
    100 105 110
    Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
    115 120 125
    Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
    130 135 140
    Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
    145 150 155 160
    Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
    165 170 175
    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
    180 185 190
    Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
    195 200 205
    Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
    210 215 220
    Ser Pro Gly Lys Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg
    225 230 235 240
    Gln Trp Leu Ala Ala Arg Ala
    245
    <210> SEQ ID NO 7
    <211> LENGTH: 861
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Fc-TMP-TMP
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (39)..(842)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 7
    tctagatttg ttttaactaa ttaaaggagg aataacat atg gac aaa act cac aca 56
    Met Asp Lys Thr His Thr
    1 5
    tgt cca cct tgt cca gct ccg gaa ctc ctg ggg gga ccg tca gtc ttc 104
    Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
    10 15 20
    ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 152
    Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
    25 30 35
    gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 200
    Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
    40 45 50
    aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 248
    Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
    55 60 65 70
    aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc 296
    Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
    75 80 85
    ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc 344
    Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
    90 95 100
    aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc 392
    Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
    105 110 115
    aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca 440
    Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
    120 125 130
    tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc 488
    Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
    135 140 145 150
    aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 536
    Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
    155 160 165
    cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 584
    Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
    170 175 180
    ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg 632
    Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
    185 190 195
    cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac 680
    Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
    200 205 210
    aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa ggt gga 728
    Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly
    215 220 225 230
    ggt ggt ggt atc gaa ggt ccg act ctg cgt cag tgg ctg gct gct cgt 776
    Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg
    235 240 245
    gct ggt ggt gga ggt ggc ggc gga ggt att gag ggc cca acc ctt cgc 824
    Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg
    250 255 260
    caa tgg ctt gca gca cgc gcataatctc gaggatccg 861
    Gln Trp Leu Ala Ala Arg
    265
    <210> SEQ ID NO 8
    <211> LENGTH: 268
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Fc-TMP-TMP
    <400> SEQUENCE: 8
    Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
    1 5 10 15
    Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
    20 25 30
    Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
    35 40 45
    His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
    50 55 60
    Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
    65 70 75 80
    Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
    85 90 95
    Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
    100 105 110
    Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
    115 120 125
    Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
    130 135 140
    Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
    145 150 155 160
    Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
    165 170 175
    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
    180 185 190
    Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
    195 200 205
    Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
    210 215 220
    Ser Pro Gly Lys Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg
    225 230 235 240
    Gln Trp Leu Ala Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly Gly Ile
    245 250 255
    Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg
    260 265
    <210> SEQ ID NO 9
    <211> LENGTH: 855
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TMP-TMP-Fc
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (39)..(845)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 9
    tctagatttg ttttaactaa ttaaaggagg aataacat atg atc gaa ggt ccg act 56
    Met Ile Glu Gly Pro Thr
    1 5
    ctg cgt cag tgg ctg gct gct cgt gct ggc ggt ggt ggc gga ggg ggt 104
    Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly Gly Gly Gly Gly Gly
    10 15 20
    ggc att gag ggc cca acc ctt cgc caa tgg ctt gca gca cgc gca ggg 152
    Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly
    25 30 35
    gga ggc ggt ggg gac aaa act cac aca tgt cca cct tgc cca gca cct 200
    Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
    40 45 50
    gaa ctc ctg ggg gga ccg tca gtt ttc ctc ttc ccc cca aaa ccc aag 248
    Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
    55 60 65 70
    gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg 296
    Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
    75 80 85
    gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac 344
    Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
    90 95 100
    ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac 392
    Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
    105 110 115
    aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 440
    Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
    120 125 130
    tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc 488
    Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
    135 140 145 150
    cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 536
    Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
    155 160 165
    gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag 584
    Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
    170 175 180
    aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac 632
    Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
    185 190 195
    atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 680
    Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
    200 205 210
    acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 728
    Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
    215 220 225 230
    aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca 776
    Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
    235 240 245
    tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc 824
    Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
    250 255 260
    ctc tcc ctg tct ccg ggt aaa taatggatcc 855
    Leu Ser Leu Ser Pro Gly Lys
    265
    <210> SEQ ID NO 10
    <211> LENGTH: 269
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TMP-TMP-Fc
    <400> SEQUENCE: 10
    Met Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly
    1 5 10 15
    Gly Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp
    20 25 30
    Leu Ala Ala Arg Ala Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys
    35 40 45
    Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
    50 55 60
    Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
    65 70 75 80
    Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
    85 90 95
    Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
    100 105 110
    Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
    115 120 125
    Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
    130 135 140
    Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
    145 150 155 160
    Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
    165 170 175
    Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
    180 185 190
    Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
    195 200 205
    Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
    210 215 220
    Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
    225 230 235 240
    Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
    245 250 255
    His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
    260 265
    <210> SEQ ID NO 11
    <211> LENGTH: 789
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TMP-Fc
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (39)..(779)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 11
    tctagatttg ttttaactaa ttaaaggagg aataacat atg atc gaa ggt ccg act 56
    Met Ile Glu Gly Pro Thr
    1 5
    ctg cgt cag tgg ctg gct gct cgt gct ggt gga ggc ggt ggg gac aaa 104
    Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly Gly Gly Gly Asp Lys
    10 15 20
    act cac aca tgt cca cct tgc cca gca cct gaa ctc ctg ggg gga ccg 152
    Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
    25 30 35
    tca gtt ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc 200
    Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
    40 45 50
    cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac 248
    Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
    55 60 65 70
    cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat 296
    Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
    75 80 85
    gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg 344
    Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
    90 95 100
    gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag 392
    Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
    105 110 115
    tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa 440
    Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
    120 125 130
    acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc 488
    Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
    135 140 145 150
    ctg ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc 536
    Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
    155 160 165
    tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag 584
    Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
    170 175 180
    agc aat ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg 632
    Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
    185 190 195
    gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag 680
    Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
    200 205 210
    agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag 728
    Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
    215 220 225 230
    gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt 776
    Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
    235 240 245
    aaa taatggatcc 789
    Lys
    <210> SEQ ID NO 12
    <211> LENGTH: 247
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TMP-Fc
    <400> SEQUENCE: 12
    Met Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly
    1 5 10 15
    Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
    20 25 30
    Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
    35 40 45
    Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
    50 55 60
    Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
    65 70 75 80
    Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
    85 90 95
    Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
    100 105 110
    Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
    115 120 125
    Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
    130 135 140
    Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
    145 150 155 160
    Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
    165 170 175
    Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
    180 185 190
    Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
    195 200 205
    Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
    210 215 220
    Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
    225 230 235 240
    Leu Ser Leu Ser Pro Gly Lys
    245
    <210> SEQ ID NO 13
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TMP
    <400> SEQUENCE: 13
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala
    1 5 10
    <210> SEQ ID NO 14
    <211> LENGTH: 36
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TMP-TMP
    <400> SEQUENCE: 14
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly
    1 5 10 15
    Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu
    20 25 30
    Ala Ala Arg Ala
    35
    <210> SEQ ID NO 15
    <211> LENGTH: 812
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Fc-EMP
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (39)..(797)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 15
    tctagatttg ttttaactaa ttaaaggagg aataacat atg gac aaa act cac aca 56
    Met Asp Lys Thr His Thr
    1 5
    tgt cca cct tgt cca gct ccg gaa ctc ctg ggg gga ccg tca gtc ttc 104
    Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
    10 15 20
    ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 152
    Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
    25 30 35
    gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 200
    Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
    40 45 50
    aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 248
    Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
    55 60 65 70
    aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc 296
    Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
    75 80 85
    ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc 344
    Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
    90 95 100
    aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc 392
    Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
    105 110 115
    aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca 440
    Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
    120 125 130
    tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc 488
    Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
    135 140 145 150
    aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 536
    Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
    155 160 165
    cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 584
    Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
    170 175 180
    ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg 632
    Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
    185 190 195
    cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac 680
    Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
    200 205 210
    aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa ggt gga 728
    Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly
    215 220 225 230
    ggt ggt ggt gga ggt act tac tct tgc cac ttc ggc ccg ctg act tgg 776
    Gly Gly Gly Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp
    235 240 245
    gtt tgc aaa ccg cag ggt ggt taatctcgtg gatcc 812
    Val Cys Lys Pro Gln Gly Gly
    250
    <210> SEQ ID NO 16
    <211> LENGTH: 253
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Fc-EMP
    <400> SEQUENCE: 16
    Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
    1 5 10 15
    Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
    20 25 30
    Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
    35 40 45
    His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
    50 55 60
    Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
    65 70 75 80
    Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
    85 90 95
    Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
    100 105 110
    Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
    115 120 125
    Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
    130 135 140
    Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
    145 150 155 160
    Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
    165 170 175
    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
    180 185 190
    Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
    195 200 205
    Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
    210 215 220
    Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Thr Tyr Ser Cys His
    225 230 235 240
    Phe Gly Pro Leu Thr Trp Val Cys Lys Pro Gln Gly Gly
    245 250
    <210> SEQ ID NO 17
    <211> LENGTH: 807
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EMP-Fc
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (39)..(797)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 17
    tctagatttg ttttaactaa ttaaaggagg aataacat atg gga ggt act tac tct 56
    Met Gly Gly Thr Tyr Ser
    1 5
    tgc cac ttc ggc ccg ctg act tgg gta tgt aag cca caa ggg ggt ggg 104
    Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys Pro Gln Gly Gly Gly
    10 15 20
    gga ggc ggg ggg gac aaa act cac aca tgt cca cct tgc cca gca cct 152
    Gly Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
    25 30 35
    gaa ctc ctg ggg gga ccg tca gtt ttc ctc ttc ccc cca aaa ccc aag 200
    Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
    40 45 50
    gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg 248
    Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
    55 60 65 70
    gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac 296
    Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
    75 80 85
    ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac 344
    Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
    90 95 100
    aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 392
    Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
    105 110 115
    tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc 440
    Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
    120 125 130
    cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 488
    Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
    135 140 145 150
    gaa cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg acc aag 536
    Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
    155 160 165
    aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac 584
    Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
    170 175 180
    atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 632
    Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
    185 190 195
    acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 680
    Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
    200 205 210
    aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca 728
    Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
    215 220 225 230
    tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc 776
    Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
    235 240 245
    ctc tcc ctg tct ccg ggt aaa taatggatcc 807
    Leu Ser Leu Ser Pro Gly Lys
    250
    <210> SEQ ID NO 18
    <211> LENGTH: 253
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EMP-Fc
    <400> SEQUENCE: 18
    Met Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys
    1 5 10 15
    Lys Pro Gln Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr His Thr Cys
    20 25 30
    Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
    35 40 45
    Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
    50 55 60
    Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
    65 70 75 80
    Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
    85 90 95
    Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
    100 105 110
    Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
    115 120 125
    Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
    130 135 140
    Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
    145 150 155 160
    Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
    165 170 175
    Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
    180 185 190
    Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
    195 200 205
    Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
    210 215 220
    Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
    225 230 235 240
    His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
    245 250
    <210> SEQ ID NO 19
    <211> LENGTH: 881
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EMP-EMP-Fc
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (41)..(871)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 19
    tctagatttg agttttaact tttagaagga ggaataaaat atg gga ggt act tac 55
    Met Gly Gly Thr Tyr
    1 5
    tct tgc cac ttc ggc cca ctg act tgg gtt tgc aaa ccg cag ggt ggc 103
    Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys Pro Gln Gly Gly
    10 15 20
    ggc ggc ggc ggc ggt ggt acc tat tcc tgt cat ttt ggc ccg ctg acc 151
    Gly Gly Gly Gly Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr
    25 30 35
    tgg gta tgt aag cca caa ggg ggt ggg gga ggc ggg ggg gac aaa act 199
    Trp Val Cys Lys Pro Gln Gly Gly Gly Gly Gly Gly Gly Asp Lys Thr
    40 45 50
    cac aca tgt cca cct tgc cca gca cct gaa ctc ctg ggg gga ccg tca 247
    His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
    55 60 65
    gtt ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg 295
    Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
    70 75 80 85
    acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct 343
    Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
    90 95 100
    gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc 391
    Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
    105 110 115
    aag aca aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc 439
    Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
    120 125 130
    agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac 487
    Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
    135 140 145
    aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc 535
    Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
    150 155 160 165
    atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg 583
    Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
    170 175 180
    ccc cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc 631
    Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
    185 190 195
    ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc 679
    Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
    200 205 210
    aat ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac 727
    Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
    215 220 225
    tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc 775
    Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
    230 235 240 245
    agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct 823
    Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
    250 255 260
    ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa 871
    Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
    265 270 275
    taatggatcc 881
    <210> SEQ ID NO 20
    <211> LENGTH: 277
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EMP-EMP-Fc
    <400> SEQUENCE: 20
    Met Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys
    1 5 10 15
    Lys Pro Gln Gly Gly Gly Gly Gly Gly Gly Gly Thr Tyr Ser Cys His
    20 25 30
    Phe Gly Pro Leu Thr Trp Val Cys Lys Pro Gln Gly Gly Gly Gly Gly
    35 40 45
    Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
    50 55 60
    Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
    65 70 75 80
    Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
    85 90 95
    Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
    100 105 110
    Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
    115 120 125
    Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
    130 135 140
    Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
    145 150 155 160
    Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
    165 170 175
    Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
    180 185 190
    Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
    195 200 205
    Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
    210 215 220
    Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
    225 230 235 240
    Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
    245 250 255
    Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
    260 265 270
    Leu Ser Pro Gly Lys
    275
    <210> SEQ ID NO 21
    <211> LENGTH: 885
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Fc-EMP-EMP
    <220> FEATURE:
    <221> NAME/KEY: CDS
    <222> LOCATION: (39)..(869)
    <223> OTHER INFORMATION:
    <400> SEQUENCE: 21
    tctagatttg ttttaactaa ttaaaggagg aataacat atg gac aaa act cac aca 56
    Met Asp Lys Thr His Thr
    1 5
    tgt cca cct tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtt ttc 104
    Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
    10 15 20
    ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct 152
    Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
    25 30 35
    gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc 200
    Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
    40 45 50
    aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca 248
    Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
    55 60 65 70
    aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc 296
    Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
    75 80 85
    ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc 344
    Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
    90 95 100
    aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc 392
    Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
    105 110 115
    aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc ctg cct cca 440
    Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
    120 125 130
    tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg gtc 488
    Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
    135 140 145 150
    aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg 536
    Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
    155 160 165
    cag ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac 584
    Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
    170 175 180
    ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aag agc agg tgg 632
    Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
    185 190 195
    cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac 680
    Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
    200 205 210
    aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa ggt gga 728
    Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly
    215 220 225 230
    ggt ggt ggc gga ggt act tac tct tgc cac ttc ggc cca ctg act tgg 776
    Gly Gly Gly Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp
    235 240 245
    gtt tgc aaa ccg cag ggt ggc ggc ggc ggc ggc ggt ggt acc tat tcc 824
    Val Cys Lys Pro Gln Gly Gly Gly Gly Gly Gly Gly Gly Thr Tyr Ser
    250 255 260
    tgt cat ttt ggc ccg ctg acc tgg gta tgt aag cca caa ggg ggt 869
    Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys Pro Gln Gly Gly
    265 270 275
    taatctcgag gatcca 885
    <210> SEQ ID NO 22
    <211> LENGTH: 277
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: Fc-EMP-EMP
    <400> SEQUENCE: 22
    Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
    1 5 10 15
    Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
    20 25 30
    Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
    35 40 45
    His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
    50 55 60
    Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
    65 70 75 80
    Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
    85 90 95
    Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
    100 105 110
    Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
    115 120 125
    Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
    130 135 140
    Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
    145 150 155 160
    Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
    165 170 175
    Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
    180 185 190
    Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
    195 200 205
    Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
    210 215 220
    Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Thr Tyr Ser Cys His
    225 230 235 240
    Phe Gly Pro Leu Thr Trp Val Cys Lys Pro Gln Gly Gly Gly Gly Gly
    245 250 255
    Gly Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys
    260 265 270
    Lys Pro Gln Gly Gly
    275
    <210> SEQ ID NO 23
    <211> LENGTH: 1546
    <212> TYPE: DNA
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: pAMG21
    <400> SEQUENCE: 23
    gcgtaacgta tgcatggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa 60
    cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct 120
    ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca acggcccgga 180
    gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca gaaggccatc 240
    ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct aaatacattc 300
    aaatatggac gtcgtactta acttttaaag tatgggcaat caattgctcc tgttaaaatt 360
    gctttagaaa tactttggca gcggtttgtt gtattgagtt tcatttgcgc attggttaaa 420
    tggaaagtga ccgtgcgctt actacagcct aatatttttg aaatatccca agagcttttt 480
    ccttcgcatg cccacgctaa acattctttt tctcttttgg ttaaatcgtt gtttgattta 540
    ttatttgcta tatttatttt tcgataatta tcaactagag aaggaacaat taatggtatg 600
    ttcatacacg catgtaaaaa taaactatct atatagttgt ctttctctga atgtgcaaaa 660
    ctaagcattc cgaagccatt attagcagta tgaataggga aactaaaccc agtgataaga 720
    cctgatgatt tcgcttcttt aattacattt ggagattttt tatttacagc attgttttca 780
    aatatattcc aattaatcgg tgaatgattg gagttagaat aatctactat aggatcatat 840
    tttattaaat tagcgtcatc ataatattgc ctccattttt tagggtaatt atccagaatt 900
    gaaatatcag atttaaccat agaatgagga taaatgatcg cgagtaaata atattcacaa 960
    tgtaccattt tagtcatatc agataagcat tgattaatat cattattgct tctacaggct 1020
    ttaattttat taattattct gtaagtgtcg tcggcattta tgtctttcat acccatctct 1080
    ttatccttac ctattgtttg tcgcaagttt tgcgtgttat atatcattaa aacggtaata 1140
    gattgacatt tgattctaat aaattggatt tttgtcacac tattatatcg cttgaaatac 1200
    aattgtttaa cataagtacc tgtaggatcg tacaggttta cgcaagaaaa tggtttgtta 1260
    tagtcgatta atcgatttga ttctagattt gttttaacta attaaaggag gaataacata 1320
    tggttaacgc gttggaattc gagctcacta gtgtcgacct gcagggtacc atggaagctt 1380
    actcgaggat ccgcggaaag aagaagaaga agaagaaagc ccgaaaggaa gctgagttgg 1440
    ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga 1500
    ggggtttttt gctgaaagga ggaaccgctc ttcacgctct tcacgc 1546
    <210> SEQ ID NO 24
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 24
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Lys Ala
    1 5 10
    <210> SEQ ID NO 25
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 25
    Ile Glu Gly Pro Thr Leu Arg Glu Trp Leu Ala Ala Arg Ala
    1 5 10
    <210> SEQ ID NO 26
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (14)..(14)
    <223> OTHER INFORMATION: At position 14, amino acid linker to an
    identical sequence
    <400> SEQUENCE: 26
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala
    1 5 10
    <210> SEQ ID NO 27
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (14)..(14)
    <223> OTHER INFORMATION: At position 14, amino acid linker to an
    identical sequence
    <400> SEQUENCE: 27
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Lys Ala
    1 5 10
    <210> SEQ ID NO 28
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (9)..(9)
    <223> OTHER INFORMATION: At position 9 disulfide linkage to position 9
    of an identical sequence
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (14)..(14)
    <223> OTHER INFORMATION: At position 14, amino acid linker to an
    identical sequence
    <400> SEQUENCE: 28
    Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala
    1 5 10
    <210> SEQ ID NO 29
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (16)..(16)
    <223> OTHER INFORMATION: Position 16 bromoacetyl group linked to
    sidechain
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (14)..(14)
    <223> OTHER INFORMATION: At position 14, amino acid linker attached
    N-to-C to Lys and to another linker and an identical sequence
    <400> SEQUENCE: 29
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala
    1 5 10
    <210> SEQ ID NO 30
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (16)..(16)
    <223> OTHER INFORMATION: Position 16 polyethylene glycol linked to
    sidechain
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (14)..(14)
    <223> OTHER INFORMATION: At position 14, amino acid linker attached
    N-to-C to Lys and to another linker and an identical sequence
    <400> SEQUENCE: 30
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala
    1 5 10
    <210> SEQ ID NO 31
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (9)..(9)
    <223> OTHER INFORMATION: Position 9 disulfide bond to residue 9 of a
    separate identical sequence
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (14)..(14)
    <223> OTHER INFORMATION: At position 14, amino acid linker to an
    identical sequence
    <400> SEQUENCE: 31
    Ile Glu Gly Pro Thr Leu Arg Gln Cys Leu Ala Ala Arg Ala
    1 5 10
    <210> SEQ ID NO 32
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (14)..(14)
    <223> OTHER INFORMATION: At position 14, amino acid linker attachment
    site
    <400> SEQUENCE: 32
    Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala
    1 5 10
    <210> SEQ ID NO 33
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (6, 7 and)..(8)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 33
    Val Arg Asp Gln Ile Xaa Xaa Xaa Leu
    1 5
    <210> SEQ ID NO 34
    <211> LENGTH: 6
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 34
    Thr Leu Arg Glu Trp Leu
    1 5
    <210> SEQ ID NO 35
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 35
    Gly Arg Val Arg Asp Gln Val Ala Gly Trp
    1 5 10
    <210> SEQ ID NO 36
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 36
    Gly Arg Val Lys Asp Gln Ile Ala Gln Leu
    1 5 10
    <210> SEQ ID NO 37
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 37
    Gly Val Arg Asp Gln Val Ser Trp Ala Leu
    1 5 10
    <210> SEQ ID NO 38
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 38
    Glu Ser Val Arg Glu Gln Val Met Lys Tyr
    1 5 10
    <210> SEQ ID NO 39
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 39
    Ser Val Arg Ser Gln Ile Ser Ala Ser Leu
    1 5 10
    <210> SEQ ID NO 40
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 40
    Gly Val Arg Glu Thr Val Tyr Arg His Met
    1 5 10
    <210> SEQ ID NO 41
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 41
    Gly Val Arg Glu Val Ile Val Met His Met Leu
    1 5 10
    <210> SEQ ID NO 42
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 42
    Gly Arg Val Arg Asp Gln Ile Trp Ala Ala Leu
    1 5 10
    <210> SEQ ID NO 43
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 43
    Ala Gly Val Arg Asp Gln Ile Leu Ile Trp Leu
    1 5 10
    <210> SEQ ID NO 44
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 44
    Gly Arg Val Arg Asp Gln Ile Met Leu Ser Leu
    1 5 10
    <210> SEQ ID NO 45
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (8)..(10)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 45
    Gly Arg Val Arg Asp Gln Ile Xaa Xaa Xaa Leu
    1 5 10
    <210> SEQ ID NO 46
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 46
    Cys Thr Leu Arg Gln Trp Leu Gln Gly Cys
    1 5 10
    <210> SEQ ID NO 47
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 47
    Cys Thr Leu Gln Glu Phe Leu Glu Gly Cys
    1 5 10
    <210> SEQ ID NO 48
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 48
    Cys Thr Arg Thr Glu Trp Leu His Gly Cys
    1 5 10
    <210> SEQ ID NO 49
    <211> LENGTH: 12
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 49
    Cys Thr Leu Arg Glu Trp Leu His Gly Gly Phe Cys
    1 5 10
    <210> SEQ ID NO 50
    <211> LENGTH: 12
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 50
    Cys Thr Leu Arg Glu Trp Val Phe Ala Gly Leu Cys
    1 5 10
    <210> SEQ ID NO 51
    <211> LENGTH: 13
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 51
    Cys Thr Leu Arg Gln Trp Leu Ile Leu Leu Gly Met Cys
    1 5 10
    <210> SEQ ID NO 52
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 52
    Cys Thr Leu Ala Glu Phe Leu Ala Ser Gly Val Glu Gln Cys
    1 5 10
    <210> SEQ ID NO 53
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 53
    Cys Ser Leu Gln Glu Phe Leu Ser His Gly Gly Tyr Val Cys
    1 5 10
    <210> SEQ ID NO 54
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 54
    Cys Thr Leu Arg Glu Phe Leu Asp Pro Thr Thr Ala Val Cys
    1 5 10
    <210> SEQ ID NO 55
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 55
    Cys Thr Leu Lys Glu Trp Leu Val Ser His Glu Val Trp Cys
    1 5 10
    <210> SEQ ID NO 56
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (8)..(9)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 56
    Cys Thr Leu Arg Glu Trp Leu Xaa Xaa Cys
    1 5 10
    <210> SEQ ID NO 57
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (8)..(10)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 57
    Cys Thr Leu Arg Glu Trp Leu Xaa Xaa Xaa Cys
    1 5 10
    <210> SEQ ID NO 58
    <211> LENGTH: 12
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (8)..(11)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 58
    Cys Thr Leu Arg Glu Trp Leu Xaa Xaa Xaa Xaa Cys
    1 5 10
    <210> SEQ ID NO 59
    <211> LENGTH: 13
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (8)..(12)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 59
    Cys Thr Leu Arg Glu Trp Leu Xaa Xaa Xaa Xaa Xaa Cys
    1 5 10
    <210> SEQ ID NO 60
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (8)..(13)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 60
    Cys Thr Leu Arg Glu Trp Leu Xaa Xaa Xaa Xaa Xaa Xaa Cys
    1 5 10
    <210> SEQ ID NO 61
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 61
    Arg Glu Gly Pro Thr Leu Arg Gln Trp Met
    1 5 10
    <210> SEQ ID NO 62
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 62
    Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala
    1 5 10
    <210> SEQ ID NO 63
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 63
    Glu Arg Gly Pro Phe Trp Ala Lys Ala Cys
    1 5 10
    <210> SEQ ID NO 64
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 64
    Arg Glu Gly Pro Arg Cys Val Met Trp Met
    1 5 10
    <210> SEQ ID NO 65
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 65
    Cys Gly Thr Glu Gly Pro Thr Leu Ser Thr Trp Leu Asp Cys
    1 5 10
    <210> SEQ ID NO 66
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 66
    Cys Glu Gln Asp Gly Pro Thr Leu Leu Glu Trp Leu Lys Cys
    1 5 10
    <210> SEQ ID NO 67
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 67
    Cys Glu Leu Val Gly Pro Ser Leu Met Ser Trp Leu Thr Cys
    1 5 10
    <210> SEQ ID NO 68
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 68
    Cys Leu Thr Gly Pro Phe Val Thr Gln Trp Leu Tyr Glu Cys
    1 5 10
    <210> SEQ ID NO 69
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 69
    Cys Arg Ala Gly Pro Thr Leu Leu Glu Trp Leu Thr Leu Cys
    1 5 10
    <210> SEQ ID NO 70
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 70
    Cys Ala Asp Gly Pro Thr Leu Arg Glu Trp Ile Ser Phe Cys
    1 5 10
    <210> SEQ ID NO 71
    <211> LENGTH: 13
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2 )..(12)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 71
    Cys Xaa Glu Gly Pro Thr Leu Arg Glu Trp Leu Xaa Cys
    1 5 10
    <210> SEQ ID NO 72
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2, 3 )..(13)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 72
    Cys Xaa Xaa Glu Gly Pro Thr Leu Arg Glu Trp Leu Xaa Cys
    1 5 10
    <210> SEQ ID NO 73
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2, 12 )..(13)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 73
    Cys Xaa Glu Gly Pro Thr Leu Arg Glu Trp Leu Xaa Xaa Cys
    1 5 10
    <210> SEQ ID NO 74
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2, 3, 13 )..(14)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 74
    Cys Xaa Xaa Glu Gly Pro Thr Leu Arg Glu Trp Leu Xaa Xaa Cys
    1 5 10 15
    <210> SEQ ID NO 75
    <211> LENGTH: 16
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 75
    Gly Gly Cys Thr Leu Arg Glu Trp Leu His Gly Gly Phe Cys Gly Gly
    1 5 10 15
    <210> SEQ ID NO 76
    <211> LENGTH: 18
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 76
    Gly Gly Cys Ala Asp Gly Pro Thr Leu Arg Glu Trp Ile Ser Phe Cys
    1 5 10 15
    Gly Gly
    <210> SEQ ID NO 77
    <211> LENGTH: 19
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 77
    Gly Asn Ala Asp Gly Pro Thr Leu Arg Gln Trp Leu Glu Gly Arg Arg
    1 5 10 15
    Pro Lys Asn
    <210> SEQ ID NO 78
    <211> LENGTH: 19
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 78
    Leu Ala Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu His Gly Asn Gly
    1 5 10 15
    Arg Asp Thr
    <210> SEQ ID NO 79
    <211> LENGTH: 19
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 79
    His Gly Arg Val Gly Pro Thr Leu Arg Glu Trp Lys Thr Gln Val Ala
    1 5 10 15
    Thr Lys Lys
    <210> SEQ ID NO 80
    <211> LENGTH: 18
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 80
    Thr Ile Lys Gly Pro Thr Leu Arg Gln Trp Leu Lys Ser Arg Glu His
    1 5 10 15
    Thr Ser
    <210> SEQ ID NO 81
    <211> LENGTH: 18
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 81
    Ile Ser Asp Gly Pro Thr Leu Lys Glu Trp Leu Ser Val Thr Arg Gly
    1 5 10 15
    Ala Ser
    <210> SEQ ID NO 82
    <211> LENGTH: 18
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TPO MIMETIC PEPTIDE
    <400> SEQUENCE: 82
    Ser Ile Glu Gly Pro Thr Leu Arg Glu Trp Leu Thr Ser Arg Thr Pro
    1 5 10 15
    His Ser
    <210> SEQ ID NO 83
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2, 4, 5, 8, 11 )..(13)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 83
    Tyr Xaa Cys Xaa Xaa Gly Pro Xaa Thr Trp Xaa Cys Xaa Pro
    1 5 10
    <210> SEQ ID NO 84
    <211> LENGTH: 28
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2, 4, 5, 8, 11, 13, 16, 18, 19, 22, 25 )..(27)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 84
    Tyr Xaa Cys Xaa Xaa Gly Pro Xaa Thr Trp Xaa Cys Xaa Pro Tyr Xaa
    1 5 10 15
    Cys Xaa Xaa Gly Pro Xaa Thr Trp Xaa Cys Xaa Pro
    20 25
    <210> SEQ ID NO 85
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (14)..(14)
    <223> OTHER INFORMATION: At position 14, amino acid linker to an
    identical sequence
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2, 4, 5, 8, 11)..(13)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 85
    Tyr Xaa Cys Xaa Xaa Gly Pro Xaa Thr Trp Xaa Cys Xaa Pro
    1 5 10
    <210> SEQ ID NO 86
    <211> LENGTH: 14
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2, 4, 5, 8, 11 )..(13)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 86
    Tyr Xaa Cys Xaa Xaa Gly Pro Xaa Thr Trp Xaa Cys Xaa Pro
    1 5 10
    <210> SEQ ID NO 87
    <211> LENGTH: 20
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 87
    Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Gln Gly Gly
    20
    <210> SEQ ID NO 88
    <211> LENGTH: 20
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 88
    Gly Gly Asp Tyr His Cys Arg Met Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Leu Gly Gly
    20
    <210> SEQ ID NO 89
    <211> LENGTH: 20
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 89
    Gly Gly Val Tyr Ala Cys Arg Met Gly Pro Ile Thr Trp Val Cys Ser
    1 5 10 15
    Pro Leu Gly Gly
    20
    <210> SEQ ID NO 90
    <211> LENGTH: 20
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 90
    Val Gly Asn Tyr Met Cys His Phe Gly Pro Ile Thr Trp Val Cys Arg
    1 5 10 15
    Pro Gly Gly Gly
    20
    <210> SEQ ID NO 91
    <211> LENGTH: 20
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 91
    Gly Gly Leu Tyr Leu Cys Arg Phe Gly Pro Val Thr Trp Asp Cys Gly
    1 5 10 15
    Tyr Lys Gly Gly
    20
    <210> SEQ ID NO 92
    <211> LENGTH: 40
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 92
    Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Gln Gly Gly Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr
    20 25 30
    Trp Val Cys Lys Pro Gln Gly Gly
    35 40
    <210> SEQ ID NO 93
    <211> LENGTH: 20
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (20)..(20)
    <223> OTHER INFORMATION: Position 20, amino acid linker to an identical
    sequence
    <400> SEQUENCE: 93
    Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Gln Gly Gly
    20
    <210> SEQ ID NO 94
    <211> LENGTH: 23
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 94
    Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Gln Gly Gly Ser Ser Lys
    20
    <210> SEQ ID NO 95
    <211> LENGTH: 46
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <400> SEQUENCE: 95
    Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Gln Gly Gly Ser Ser Lys Gly Gly Thr Tyr Ser Cys His Phe Gly
    20 25 30
    Pro Leu Thr Trp Val Cys Lys Pro Gln Gly Gly Ser Ser Lys
    35 40 45
    <210> SEQ ID NO 96
    <211> LENGTH: 23
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (23)..(23)
    <223> OTHER INFORMATION: Position 23, amino acid linker to an identical
    sequence
    <400> SEQUENCE: 96
    Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Gln Gly Gly Ser Ser Lys
    20
    <210> SEQ ID NO 97
    <211> LENGTH: 22
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (22)..(22)
    <223> OTHER INFORMATION: Position 22 linked through epsilon amine to
    lysyl, which is linked to a separate identical sequence through
    that sequence′s alpha amine
    <400> SEQUENCE: 97
    Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Gln Gly Gly Ser Ser
    20
    <210> SEQ ID NO 98
    <211> LENGTH: 23
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (23)..(23)
    <223> OTHER INFORMATION: At position 23 biotin linked to the sidechain
    through a linker
    <400> SEQUENCE: 98
    Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys
    1 5 10 15
    Pro Gln Gly Gly Ser Ser Lys
    20
    <210> SEQ ID NO 99
    <211> LENGTH: 5
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: G-CSF-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (4)..(4)
    <223> OTHER INFORMATION: At position 4 disulfide bond to residue 4 of a
    separate identical sequence
    <400> SEQUENCE: 99
    Glu Glu Asp Cys Lys
    1 5
    <210> SEQ ID NO 100
    <211> LENGTH: 5
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: G-CSF-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (4)..(4)
    <223> OTHER INFORMATION: At position 4, Xaa is an isoteric ethylene
    spacer linked to a separate identical sequence
    <400> SEQUENCE: 100
    Glu Glu Asp Xaa Lys
    1 5
    <210> SEQ ID NO 101
    <211> LENGTH: 6
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: G-CSF-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1)..(1)
    <223> OTHER INFORMATION: Position 1, Xaa is a pyroglutamic acid residue
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (5)..(5)
    <223> OTHER INFORMATION: Position 5, Xaa is an isoteric ethylene spacer
    linked to a separate identical sequence.
    <400> SEQUENCE: 101
    Xaa Gly Glu Asp Xaa Lys
    1 5
    <210> SEQ ID NO 102
    <211> LENGTH: 5
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: G-CSF-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1)..(1)
    <223> OTHER INFORMATION: Position 1, Xaa is a picolinic acid residue
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (4)..(4)
    <223> OTHER INFORMATION: Position 4, Xaa is an isoteric ethylene spacer
    linked to a separate identical sequence.
    <400> SEQUENCE: 102
    Xaa Ser Asp Xaa Lys
    1 5
    <210> SEQ ID NO 103
    <211> LENGTH: 5
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: G-CSF-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (5)..(5)
    <223> OTHER INFORMATION: At position 5, amino acid linker to an
    identical sequence
    <400> SEQUENCE: 103
    Glu Glu Asp Cys Lys
    1 5
    <210> SEQ ID NO 104
    <211> LENGTH: 5
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: G-CSF-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (5)..(5)
    <223> OTHER INFORMATION: At position 5, amino acid linker to an
    identical sequence
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (4)..(4)
    <223> OTHER INFORMATION: Xaa = any amino acid
    <400> SEQUENCE: 104
    Glu Glu Asp Xaa Lys
    1 5
    <210> SEQ ID NO 105
    <211> LENGTH: 6
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: ANTIVIRAL (HBV)
    <400> SEQUENCE: 105
    Leu Leu Gly Arg Met Lys
    1 5
    <210> SEQ ID NO 106
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 106
    Tyr Cys Phe Thr Ala Ser Glu Asn His Cys Tyr
    1 5 10
    <210> SEQ ID NO 107
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 107
    Tyr Cys Phe Thr Asn Ser Glu Asn His Cys Tyr
    1 5 10
    <210> SEQ ID NO 108
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 108
    Tyr Cys Phe Thr Arg Ser Glu Asn His Cys Tyr
    1 5 10
    <210> SEQ ID NO 109
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 109
    Phe Cys Ala Ser Glu Asn His Cys Tyr
    1 5
    <210> SEQ ID NO 110
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 110
    Tyr Cys Ala Ser Glu Asn His Cys Tyr
    1 5
    <210> SEQ ID NO 111
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 111
    Phe Cys Asn Ser Glu Asn His Cys Tyr
    1 5
    <210> SEQ ID NO 112
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 112
    Phe Cys Asn Ser Glu Asn Arg Cys Tyr
    1 5
    <210> SEQ ID NO 113
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 113
    Phe Cys Asn Ser Val Glu Asn Arg Cys Tyr
    1 5 10
    <210> SEQ ID NO 114
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 114
    Tyr Cys Ser Gln Ser Val Ser Asn Asp Cys Phe
    1 5 10
    <210> SEQ ID NO 115
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 115
    Phe Cys Val Ser Asn Asp Arg Cys Tyr
    1 5
    <210> SEQ ID NO 116
    <211> LENGTH: 11
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 116
    Tyr Cys Arg Lys Glu Leu Gly Gln Val Cys Tyr
    1 5 10
    <210> SEQ ID NO 117
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 117
    Tyr Cys Lys Glu Pro Gly Gln Cys Tyr
    1 5
    <210> SEQ ID NO 118
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 118
    Tyr Cys Arg Lys Glu Met Gly Cys Tyr
    1 5
    <210> SEQ ID NO 119
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 119
    Phe Cys Arg Lys Glu Met Gly Cys Tyr
    1 5
    <210> SEQ ID NO 120
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 120
    Tyr Cys Trp Ser Gln Asn Leu Cys Tyr
    1 5
    <210> SEQ ID NO 121
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 121
    Tyr Cys Glu Leu Ser Gln Tyr Leu Cys Tyr
    1 5 10
    <210> SEQ ID NO 122
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 122
    Tyr Cys Trp Ser Gln Asn Tyr Cys Tyr
    1 5
    <210> SEQ ID NO 123
    <211> LENGTH: 9
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: TNF ANTAGONIST PEPTIDE
    <400> SEQUENCE: 123
    Tyr Cys Trp Ser Gln Tyr Leu Cys Tyr
    1 5
    <210> SEQ ID NO 124
    <211> LENGTH: 10
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: EPO-MIMETIC PEPTIDE
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (1)..(1)
    <223> OTHER INFORMATION: Xaa (Pos1) can be C, A, a-amino-g-bromobutyric
    acid or Hoc.
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (2)..(2)
    <223> OTHER INFORMATION: Xaa can be R, H, L or W.
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (3)..(3)
    <223> OTHER INFORMATION: Xaa can be M, F or I.
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (6)..(6)
    <223> OTHER INFORMATION: Xaa can be any one of the 20 L-amino acids or
    the stereoisomeric D-amino acids.
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (9)..(9)
    <223> OTHER INFORMATION: Xaa can be D, E, I, L or V.
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <222> LOCATION: (10)..(10)
    <223> OTHER INFORMATION: Xaa can be a-amino-g-bromobutyric acid or Hoc,
    provided that either Xaa (Pos1) or Xaa (Pos10) is C or Hoc.
    <400> SEQUENCE: 124
    Xaa Xaa Xaa Gly Pro Xaa Thr Trp Xaa Xaa
    1 5 10
    <210> SEQ ID NO 125
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: CTLA4-MIMETIC
    <400> SEQUENCE: 125
    Gly Phe Val Cys Ser Gly Ile Phe Ala Val Gly Val Gly Arg Cys
    1 5 10 15
    <210> SEQ ID NO 126
    <211> LENGTH: 15
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: CTLA4-MIMETIC
    <400> SEQUENCE: 126
    Ala Pro Gly Val Arg Leu Gly Cys Ala Val Leu Gly Arg Tyr Cys
    1 5 10 15
    <210> SEQ ID NO 127
    <211> LENGTH: 27
    <212> TYPE: PRT
    <213> ORGANISM: Artificial Sequence
    <220> FEATURE:
    <223> OTHER INFORMATION: C3B ANTAGONIST
    <400> SEQUENCE: 127
    Ile Cys Val Val Gln Asp Trp Gly His His Arg Cys Thr Ala Gly His
    1 5 10 15
    Met Ala Asn Leu Thr Ser His Ala Ser Ala Ile
    20 25