Modified peptides as therapeutic agents

(21) Filing number: 366080 (22) Filing date: October 25, 1999 (86) Date and number of international filing:

1999-10-25, PCT / US99 / 025044 (87) International application publication date and number:

May 4, 2000, WO00 / 24782 (11) 211164 (13) B1 (51) Int.Cl.

C07K 19/00 (2006.01) C12N 15/62 (2006.01) C12N 1/21 (2006.01) C12N 15/70 (2006.01)

Novel therapeutic agent compounds, DNA encoding these compounds, expression vectors containing the indicated DNA acids, host cells containing (54) the indicated expression vectors, a pharmaceutical composition containing the indicated novel compounds, the use of these compounds, and a method of producing new therapeutic agent compounds (30) Priority :

October 23, 1998, US, 60 / 105,371 October 22, 1999, US, 09 / 428,082 (43) Application announced:

24.01.2005 BUP 02/05 (73) The holder of the patent:

AMGEN INC., Thousand Oaks, US (72) Inventor (s):

ULRICH FEIGE, Newbury Park, US CHUAN-FA LIU, Longmont, US JANET CHEETHAM, Montecito, US

THOMAS CHARLES BOONE, Newbury Park, US (45) The grant of the patent was announced:

April 30, 2012 WUP 04/12 (74) Plenipotentiary:

item. stalemate. Ewa Malewska

PL 211 164 B1

Description of the invention

The invention relates to novel therapeutic agent compounds, as well as DNA encoding these compounds, expression vectors containing the indicated DNA acids, host cells containing the indicated expression vectors, a pharmaceutical composition containing the indicated novel compounds, the use of these compounds, and a method of producing new therapeutic agent compounds.

Background of the invention

Recombinant proteins represent a new class of therapeutic agents. Such recombinant therapeutic agents have resulted in advances in protein compositions and chemical modifications. Such modifications can protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Protein modifications may also increase therapeutic protein stability, circulation time, and biological activity. Review article describing protein modifications and fusion proteins published by Francis (1992),

Focus on Growth Factors 3: 4-10 (Mediskrypt, London) is incorporated by reference in the present application.

One useful modification is linkage to the "Fc" domain of an antibody. Antibodies include two functionally independent parts: a variable domain known as "Fab", which binds an antigen, and a constant domain known as "Fc", which binds to effector functions such as complement activation and phagocytic cell attack. Fc has a long plasma half-life while Fab has a short lifetime. Capon et al. (1989), Nature 337: 525-31. In a construct with a therapeutic protein, the Fc domain may provide an extension of half-life or add functions such as Fc receptor binding, protein A binding, complement fixation, and possibly even placental transfer. Id. Table 1 summarizes the use of Fc fusion known in the art.

Table 1 - Fc fusion with therapeutic proteins

<td>Form Fc</td><td>Fusion partner</td><td>Therapeutic indications</td><td>Reference</td>

<td>IgG1</td><td>N-last CD30-L</td><td>Hodgkin's disease; anaplastic lymphoma; T-cell leukemia</td><td>Patent description USA 5,480,981</td>

<td>Mouse FcY2a</td><td>IL-10</td><td>Anti-inflammatory; against transplant rejection</td><td>Zheng et al. (1995) J. Immunol. 154: 5590-600</td>

<td>IgG1</td><td>TNF receptor</td><td>Septic shock</td><td>Fisher et al. (1996) N. Engl. J. Med. 334: 1697-1702: Van Zee, K. et al. (1996) J. Immunol. 156: 2221-30</td>

<td>IgG, IgA, IgM, or IgE (excluding first domain)</td><td>TNF receptor</td><td>Inflammation, disturbance autoimmune</td><td>U.S. Patent 5,808,029, granted September 15, 1998</td>

<td>IgG1</td><td>CD4 receptor</td><td>AIDS</td><td>Capon et al. (1989), Nature 337: 525-31</td>

<td>IgG1, IgG3</td><td>N-last IL-2</td><td>Anti-cancer, antiviral</td><td>Harvill ijn. (1995), Immunotech. 1: 95-105</td>

<td>IgG1</td><td>C-end of OPG</td><td>Osteoarthritis; Bone Density</td><td>WO 97/23614, published July 3, 1997</td>

<td>IgG1</td><td>N-last leptin</td><td>anti-obesity</td><td>Application PCT / US 97/23183 filed on December 11, 1997</td>

<td>Human Ig Cy1</td><td>CTLA-4</td><td>Autoimmune disorders</td><td>Linsley (1991) J. Exp. Med. 174: 561-9</td>

The phage peptide library display system has proved to be an effective method of identifying such agonists and peptide antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Patent No. 5,223,409, issued June 29, 1993; U.S. Patent No. 5,733,731, issued March 31, 1998; US patent specification

PL 211 164 B1

No. 5,498,530, issued March 12, 1996; U.S. Patent No. 5,432,018, issued Jul. 11, 1995; U.S. Patent No. 5,338,665, issued Aug. 3, 1994; U.S. Patent No. 5,922,545, issued Jul 13, 1999; WO 96/40987, published December 19, 1996; and WO 98/15833, published April 16, 1998 (each of which is incorporated herein by reference). In such libraries, random peptide sequences are displayed fused to fibrous phage shell proteins. Typically, the presented peptides are isolated by affinity chromatography based on the antibody-immobilized extracellular domain of the receptor. Retained phages are enriched during successive rounds of affinity purification and repropagation. The best binding peptides can be sequenced to identify key residues in one or more structurally related peptide families. See, for example, Cwirla et al. (1997), Science 276: 1696-9, where two different families have been identified. Such peptide sequences may also suggest which residues can be safely replaced by alanine scanning or DNA mutagenesis. Mutagenesis libraries can be created and screened to further optimize the sequence of the best binding compounds. Lowman (1997), Ann. Rev. Biofis. Biomol. Struct. 26: 401-24.

Structural analysis of protein-protein interactions can also be used to suggest peptides capable of mimicking (mimicry) 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 a large protein ligand from which a peptide can be designed. See, for example, Takasaki et al. (1997), Nature Biotech. 15: 1266-70. These analytical methods can also be used to study interactions between the protein receptor and peptides selected on the basis of phage display, which may suggest further modification of the peptides to increase binding affinity.

In peptide research, other methods compete with the phage display method. And the peptide library can be fused to the carboxyl terminus of the lac repressor and expressed in E. coli. Another method using E. coli allows for presentation on the outer cell membrane by fusion with 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 stopped before the ribosomes are released, resulting in a library of polypeptides with their bound RNA still attached thereto. Hereinafter, this and related methods are collectively referred to as "ribosome presentation." Other methods include chemically linking peptides to RNA; see, for example, Roberts &amp; amp; Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303. Hereinafter, these and related methods are collectively referred to as "RNA-peptide screening." Libraries of chemically obtained peptides have been developed in which the peptides are immobilized on durable, non-biological materials such as polyethylene rods or solvent permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass plates. Hereinafter, these and related methods are collectively referred to as "peptide chemical screening." Chemical-peptide screening can be advantageous in that it allows the use of D-amino acids and other unnatural analogs as well as non-peptide elements. Both biological and chemical methods have been reviewed - in: Wells &amp; Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.

Conceptually, a mimetic peptide of any protein can be discovered using phage display or other methods discussed above. These methods have been used for epitope mapping to identify critical amino acids in protein-protein interactions, but may lead to the discovery of new therapeutic agents. For example, Cortese et al. (1996), Curr. Opin. Biotech. 7: 616-21. Peptide libraries are currently most used in immunological research such as epitope mapping. Kreeger (1996), The Scientist 10 (13): 19-20.

Of particular interest in this work is the use of a peptide library and other techniques in the discovery of pharmacologically active peptides. A number of such peptides identified in the art are summarized in Table 2. These peptides are described in the listed publications, each of which is incorporated herein by reference. The pharmacological activity of the peptides is described, and in many cases the abbreviated identification term is then given in parentheses. Some of these peptides were modified (for example to form C-terminally cross-linked dimers). Typically, peptide libraries have been screened for receptor binding to detect the pharmacological activity of the protein (for example, the EPO receptor). In at least one (CTLA4), a peptide library was screened for binding to a monoconal antibody.

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Table 2 - Pharmacologically active peptides

<td>Form of the peptide</td><td>Tie partner / considered Protein^and</td><td>Activity pharmacological</td><td>Reference</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>Interpeptide disulfide bond</td><td>EPO receptor</td><td>EPO-mimetic</td><td>Wrighton et al. (1996), Science 273: 458-63; U.S. Patent No. 5,773,569, issued Jun. 30, 1998: Wrighton et al.</td>

<td>C-final cross-linked dimer</td><td>EPO receptor</td><td>EPO-mimetic</td><td>Livnah et al. (1996), Science 273: 464-71; Wrighton et al. (1997), Nature Biotechnology 15: 1261-5; International patent application WO 96/40772, published on Dec. 19. 1996</td>

<td>Linear</td><td>EPO receptor</td><td>EPO-mimetic</td><td>Naranda et al. (1999) Proc. Natl. Acad. Sci. USA, 96: 7569-74</td>

<td>Linear</td><td>c-Mpl</td><td>TPO-mimetic</td><td>Cwirla et al. (1997) Science 276: 1696-9; U.S. Patent No. 5,869,451, issued Feb 9, 1999; US patent specification No. 5,932,946, issued August 3, 1999</td>

<td>C-terminal cross-linked dimer</td><td>c-Mpl</td><td>TPO-mimetic</td><td>Cwirla et al. (1997), Science 276: 1696-9</td>

<td>Disulfide dimer</td><td></td><td>Stimulating hematopoiesis ("G-CSF-mimetic")</td><td>Paukovits et al. (1984), Hoppe-Seylers Z. Fisiol. Chem. 365: 303-11; Laerum et al. (1988), Exp. Hemat. 16: 274-80</td>

<td>Alkylene linked dimer</td><td></td><td>G-CSF-mimetic</td><td>Bhatnagar et al. (1996), 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</td>

<td>Linear</td><td>IL-1 receptor</td><td>Inflammatory and autoimmune diseases ("IL-1 antagonist" or "IL-1ra mimetic")</td><td>U.S. Patent No. 5,608,035; U.S. Patent No. 5,786,331; U.S. Patent No. 5,880,096; Yanofsky et al. (1996), Proc. Natl. Acad. Sci. 93: 7381-6; Akeson et al. (1996), J. Biol. Chem. 271: 30517-23; Wiezorek et al. (1997), Pol. J. Farmacol. 49: 107-17; Yanofsky (1996) PNAs, 93: 7381-7386.</td>

<td>Linear</td><td>Serum Thymus Factor (FTS)^b</td><td>Lymphocyte stimulation ("FTS mimetic")</td><td>Inagaki-Ohara et al. (1996), Cellular Immunol. 171: 30-40; Yoshida (1984), Int. J. Immunofarmacol, 6: 141-6.</td>

<td>Intrinsic Peptide disulfide bond</td><td>CTLA4 MAb</td><td>CTLA4-mimetic</td><td>Fukumoto et al. (1998) Nature Biotech. 16: 267-70</td>

<td>Exocyclic</td><td>TNF-α receptor</td><td>TNF-α antagonist</td><td>Takasaki et al. (1997) Nature Biotech. 15: 1266-70; WO 98/53842, published December 3, 1998</td>

<td>Linear</td><td>TNF-α receptor</td><td>TNF-α antagonist</td><td>Chirinos-Rojas (), J. Imm., 5621-5626.</td>

<td>Intra-peptide disulfide bond</td><td>C3b</td><td>Inhibiting the activation of the complement; autoimmune diseases ("C3b antagonist")</td><td>Sahu et al. (1996), J. Immunol. 157: 884-91; Morikis et al. (1998), Protein Sci. 7: 619-27</td>

PL 211 164 B1 cont. table 2

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>Linear</td><td>Vinculin</td><td>Cell adhesion processes - cell growth, differentiation, wound healing, cancer metastasis ("vinulin binding")</td><td>Adey et al. (1997), Biochem. J. 324: 523-8</td>

<td>Linear</td><td>C4-binding protein (C4BP)</td><td>Anticoagulant</td><td>Linse et al. (1997) J. Biol. Chem. 272: 14685-65</td>

<td>Linear</td><td>urokinase receptor</td><td>Processes related to the interaction of urokinase with its receptor (for example, angiogenesis, cancer cell invasion, and metastasis); ("UKR antagonist")</td><td>Goodson et al. (1994) Proc. Natl. Acad. Sci. 91: 7129-33; International Application WO 97/35969, published October 2, 1997</td>

<td>Linear</td><td>Mdm2, Hdm2</td><td>Inhibition of p53 inactivation mediated by Mdm2 or hdm2; anti-cancer ("Mdm / hdm antagonist")</td><td>Picksley et al. (1994), Oncogene 9: 2523-9; Botteer et al. (1997) J. Mol. Biol. 269: 744-56; Bottger ijn. (1996) Oncogene 13: 2141-7</td>

<td>Linear</td><td>_p21^WAF1</td><td>Antitumor By mimicking the activity of p21^WAF1</td><td>Ball et al. (1997), Curr. Biol. 7: 71-80</td>

<td>Linear</td><td>Transferase farnesyl</td><td>anti-cancer by preventing the ras oncogene from activating</td><td>Gibbs ijn. (1994), Cell 77: 175-178</td>

<td>Linear</td><td>Ras effector domain</td><td>Anti-cancer by inhibiting the biological function of the ras oncogene</td><td>Moodie et al. (1994), Trends Genet 10: 44-48 Rodriguez et al. (1994) Nature 370: 527-532</td>

<td>Linear</td><td>domains SH2 / SH3</td><td>Anti-cancer by inhibiting tumor growth with activated tyrosine kinase</td><td>Pawson et al (1993), Curr. Biol. 3: 434-432 Yu ijn. (1994), Cell 76: 933-945</td>

<td>Linear</td><td>_p16lNK4</td><td>Anti-cancer by mimicking the activity of p16; for example by inhibiting the cyclin D-Cdk complex ("p16-mimetic")</td><td>Fahraeus et al. (1996) Curr. Biol. 6: 84-91</td>

<td>Linear</td><td>Src, Lyn</td><td>Inhibition of Mast cell activation, IgE related conditions, type I hypersensitivity ("Mast cell antagonist")</td><td>Stauffer et al. (1997) Biochem. 36: 9388-94</td>

<td>Linear</td><td>Mast cell protease</td><td>Treatment of inflammatory disorders mediated by the release of tryptase-6 ("Mast cell protease inhibitors")</td><td>International Application WO 98/33812, published August 6, 1998</td>

PL 211 164 B1 cont. table 2

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>Linear</td><td>Domemy SH3</td><td>Treatment of SH3 Mediated Conditions ("SH3 Antagonists")</td><td>Rickles et al. (1994), EMBO J. 13: 5598-5604; Sparks jjin. (1994) J. Biol. Chem. 269: 23853-6; Sparks et al. (1996), Proc. Natl. Acad. Sci. 93: 1540-4</td>

<td>Linear</td><td>HBV core antigen (HBcAg)</td><td>Treatment of HBV viral infections ("anti-HBV")</td><td>Dyson &amp; Muray (1995) Proc. Natl. Acad. Sci. 92: 2194-8</td>

<td>Linear</td><td>Selectins</td><td>Neutrophilic adhesion; inflammatory diseases ("selectin antagonist")</td><td>Martens et al. (1995) J. Biol. Chem. 270: 21129-36; European patent application EP 0 714 912, published June 5, 1996</td>

<td>linear cyclized</td><td>Calmodulin</td><td>Antagonist calmodulin</td><td>Pierce et al. (1995), Molec. Diversity 1: 259-65; Dedman et al. (1993) J. Biol. Chem. 268: 23025-30; Adey &amp; Kay (1996), Gene 169: 133-4</td>

<td>Linear, cyclized</td><td>Integrins</td><td>Targeting a tumor; treatment of conditions associated with integrin-mediated cellular events, including platelet aggregation, thrombosis, wound healing, osteoporosis, tissue regeneration, angiogenesis (for example, for treating cancer), and tumor invasion ("integrin binding")</td><td>International Application WO 95/14714, published June 1, 1995; WO 97/08203, published March 6, 1997; WO 98/10795 published March 19, 1998; WO 99/24462, published May 20, 1999; Kraft et al. (1999) J. Biol. Chem. 274: 1979-1985</td>

<td>Cyclic, linear</td><td>Fibronectin and the extracellular matrix of the components of T cells and macrophages</td><td>Treatment of inflammation and autoimmune conditions</td><td>WO 98/09985, published March 12, 1998</td>

<td>Linear</td><td>Somatostatin and cortistatin</td><td>treatment or prevention of hormone-secreting tumors, acromegaly, gigantism, dementia, gastric ulcer, tumor growth, inhibition of hormone secretion, modulation of sleep or neural activity</td><td>European patent application 0 911 393, published August 28, 1999</td>

<td>Linear</td><td>Bacterial lipopolysaccharide</td><td>antibiotic; septic shock; CAP-modulated disorders37</td><td>U.S. Patent No. 5,877,151, issued March 2, 1999</td>

<td>Linear or cyclic, including the D-amino acid</td><td>Pardaxine, melittin</td><td>Antipathogenic</td><td>WO 97/31019, published August 28, 1997</td>

<td>linear, cyclic</td><td>VIP</td><td>Impotence, neurodegenerative disorders</td><td>WO 97/40070, published October 30, 1997</td>

PL 211 164 B1 cont. table 2

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>Linear</td><td>CTLs</td><td>Cancer</td><td>EP 0 770 624, published May 2, 1997</td>

<td>Linear</td><td>THF-gamma2</td><td></td><td>Burnstein (1988), Biochem. 27: 4066-71.</td>

<td>linear</td><td>Amylin</td><td></td><td>Cooper (1987), Proc. Natl. Acad. Sci., 84: 8628-32.</td>

<td>linear</td><td>Adrenomedulin</td><td></td><td>Kitamura (1993), BBRC, 192: 553-60.</td>

<td>Cyclic, linear</td><td>VEGF</td><td>Anti-angiogenic; cancer, rheumatoid arthritis, diabetic retinopathy, psoriasis ("VEGF antagonist")</td><td>Fairbrother (1998), Biochem. 37: 17754-17764.</td>

<td>Cyclical</td><td>MMP</td><td>Inflammatory and autoimmune disorders; tumor growth ("MMP inhibitor")</td><td>Koivunen (1999), Nature Biotech., 17: 768-774.</td>

<td></td><td>fragment of HGH</td><td></td><td>U.S. Patent No. 5,869,452</td>

<td></td><td>Echistatin</td><td>Inhibition platelet aggregation</td><td>Gan (1988), J. Biol. Chem., 263: 19827-32.</td>

<td>linear</td><td>SLE autoantibody</td><td>SLE</td><td>WO 96/30057, published October 3, 1996</td>

<td></td><td>a-GD1</td><td>Tumor metastasis suppression</td><td>Ishikawa et al. (1998) FEBS Lett. 441 (1): 20-4</td>

<td></td><td>beta-2-glyco- antiphospholipid antibodies protein I (e2GPI)</td><td>Endothelial cell activation, antiphospholipid syndrome (APS), thromboembolic phenomena, thrombocytopenia, recurrent miscarriage</td><td>Blank et al. (1999) Proc. Natl. Acad. Sci. USA 96: 5164-8</td>

<td>Linear</td><td>Beta chain T cell receptor</td><td>diabetes</td><td>WO 96/11214, published April 18, 1996</td>

^and The protein listed in this column may be bound by the peptide of interest (e.g. EPO receptor, IL-1 receptor) or mimicked (mimetic) by the peptide of interest. The references listed for each of them explain whether the molecule is bound or mimated by these peptides.

^b FTS is a thymic hormone mimicked by a compound molecule of the invention rather than by its receptor binding by a compound molecule of the invention.

The peptides identified by the peptide library screen were considered leads in the development of therapeutic compounds rather than therapeutic compounds per se. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, the mechanisms of cellular cleansing in the corneal-endothelial system, or proteolytic degradation. Francis (1992), Focus on Growth Factors 3: 4-11. As a result, these identified peptides are now used in the art to validate therapeutic targets or as a scaffold to design organic compounds that might not be as easy or quick to identify by screening a chemical library. Lowman (1997), Ann. Rev. Biofis. Biomol. Struct. 26: 401-24; Kay et al. (1998), Drug Disc. Today 3: 370-8. A major contribution to the arts would be a way by which such peptides could more readily lead to therapeutic agents.

The essence of the invention

The invention relates to novel compounds that are therapeutic agents.

The invention includes compounds of formula (X¹) aF¹- (X²) b

PL 211 164 B1 and its multimers, where:

F.¹ is an Fc domain;

X¹ and X², each, is independently selected from - (L.¹) cP¹, - (L.¹) cP¹- (L.²) dP², - (L.¹) cP¹- (L.²)_dP.²- (L.³) eP³, and - (L.¹) cP¹- (L.²) dP²- (L.³) eP³- (L.⁴) fP⁴

P.¹, P², P³ and P⁴each independently represent randomized sequences of pharmacologically active peptides;

L.¹, L.², L.³ and L.⁴each independently represent a linker; aa, b, c, d, e and f are each independently 0 or 1, with the proviso that at least one of a and b is 1 and "peptide" denotes molecules of 2 to 40 amino acids, and neither X¹nor X² it is not a naturally occurring protein.

A preferred group of compounds according to the invention are in particular compounds having the structure defined by the formula

X¹-F¹ or F¹-X².

Another group of preferred compounds of the invention are compounds having the structure defined by formula F¹- (L.¹) cP¹.

A further preferred group of compounds according to the invention are compounds having the structure defined by the formula

F.¹- (L.¹) cP¹- (L.²) dP².

In the compounds according to the invention, preferably F¹ is the Fc domain of IgG.

In the compounds according to the invention, preferably F¹ is the Fc domain of IgG1.

In the compounds according to the invention, preferably F¹ includes the sequence of SEQ ID NO: 2.

Among the compounds according to the invention, a preferred group are those compounds of the above-defined formula (X¹) aF¹- (X²) b, especially those in which F¹ is the Fc domain of IgG, where P¹, P², P³ and P⁴ each independently is a randomized IL-1 antagonist peptide sequence.

Preferably, the IL-1 antagonist peptide sequence is selected from the group consisting of SEQ ID NOS: 212, 907, 908, 909, 910, 917 and 979.

Preferably also, the IL-1 antagonist peptide sequence is selected from the group consisting of SEQ ID NOS: 213 to 271, 671 to 906, 911 to 916 and 918 to 1023.

Among the compounds according to the invention, a preferred group are those compounds of the above-defined formula (X¹) aF¹- (X²) b, especially those in which F¹ is the Fc domain of IgG, where P¹, P², P³ and P⁴ each independently is a randomized EPO-mimetic peptide sequence.

Preferably, the EPO-mimetic peptide sequence is selected from Table 5 given below.

Preferably these compounds comprising a sequence selected from SEQ ID NOS: 83, 84, 85, 124, 419, 420, 421 and 461.

Preferably also, the compounds include a sequence selected from SEQ ID NOS: 339 and 340.

Compounds which have a sequence selected from SEQ ID NOS: 20 and 22 are also preferred.

Among the compounds according to the invention, a preferred group are those compounds of the above-defined formula (X¹) aF¹- (X²) b, especially those in which F¹ is the Fc domain of IgG, where P¹, P², P³ and P⁴each independently is a randomized TPO-mimetic peptide sequence.

Preferably, the sequence P¹ is a TPO-mimetic peptide sequence selected from Table 6 below.

Preferably also, compounds containing randomized TPO-mimetic peptide sequences have a sequence selected from SEQ ID NOS: 6 and 12.

Among the compounds according to the invention, a preferred group are those compounds of the above-defined formula (X¹) aF¹- (X²) b, especially those in which F¹ is the Fc domain of IgG, where P¹, P², P³ and P⁴each independently is a randomized TNF antagonist peptide sequence.

Among the compounds according to the invention, a preferred group are those compounds of the above-defined formula (X¹) aF¹- (X²) b, especially those in which F¹ is the Fc domain of IgG, where P¹, P², P³ and P⁴each independently is a randomized sequence of a GCSF-mimetic peptide.

The invention also relates to a DNA acid encoding any of the compounds of the invention as defined above.

The invention also includes an expression vector containing DNA encoding any of the compounds of the invention as defined above.

PL 211 164 B1

Also encompassed by the invention is a host cell comprising an expression vector comprising DNA encoding any of the compounds of the invention as defined above.

Preferably, the host cell of the invention is an E. coli cell.

The invention further relates to a pharmaceutical composition comprising any compound of the invention together with a pharmaceutically acceptable diluent, preservative, solubilizing agent, adjuvant and / or carrier.

Also, the use of any compound of the invention as a therapeutic or prophylactic agent is within the scope of the present invention.

Finally, the invention also relates to a process for the preparation of a pharmacologically active compound of the above-defined structure, which according to the invention is characterized in that it comprises

a) selecting at least one randomized peptide modulating the activity of the protein in question; and

b) producing a pharmacological agent comprising at least one Fc domain covalently linked to at least one amino acid sequence of the selected peptide or peptides, wherein the term "peptide" refers to molecules of 2 to 40 amino acids.

Preferably, the method of the invention uses a peptide selected by a process comprising screening a phage display library, an E. coli display library, a ribosome library, or a chemical peptide library.

Preferably, a randomized EPOmimetic peptide is used as the peptide in the method of the invention.

Preferably, a randomized TPOmimetic peptide is used as the peptide in the method of the invention.

Preferably, a randomized IL-1 antagonist is used as the peptide in the method of the invention.

Preferably, a randomized GCSF-mimetic peptide is used as the peptide in the method of the invention.

Preferably, a randomized TNF antagonist is used as the peptide in the method of the invention.

Preferably, the peptide used in the process of the invention is a peptide selected from Tables 4 to 20 below.

Preferably, the method of the invention produces a compound having the structure defined by formula (X¹) aF¹- (X²) b or its multimer, where:

F.¹ is an Fc domain;

X¹ and X²each, independently, is selected from - (L.¹) cP¹, - (L.¹) cP¹- (L.²) dP², - (L.¹) cP¹- (L.²)_dP.²- (L.³) eP³ and - (L.¹) cP¹- (L.²) dP²- (L.³) e -P³- (L.⁴) fP⁴

P.¹, P², P³ and P⁴each independently represent randomized sequences of pharmacologically active peptides;

L.¹, L.², L.³ and L.⁴each independently is a linker; aa, b, c, d, e and f each independently represent "0" or "1" with the proviso that at least one of "a" and "b" represents "1" and neither X¹nor X² it is not a naturally occurring protein.

Preferably, the method of the invention produces a compound having a structure as defined by the formula

X¹-F¹ or

F.¹-X².

Preferably, the method of the invention produces a compound having a structure as defined by the formula

F.¹- (L.¹) cP¹ or

F.¹- (L.¹) cP¹- (L.²) dP².

Preferably, in the method according to the invention, as an Fc domain, also defined by the symbol F in the above formulas¹ the Fc domain of IgG is used.

PL 211 164 B1

Preferably, in the method according to the invention, as an Fc domain, also defined by the symbol F in the above formulas¹ the Fc domain of IgG1 is used.

Preferably, in the method according to the invention, as an Fc domain, also defined by the symbol F in the above formulas¹ an Fc domain comprising the sequence of SEQ ID NO: 2 is used.

The present invention relates to a method whereby the in vivo half-life of one or more biologically active peptides is increased by fusion with a carrier that is an Fc domain.

The peptides screened in step (a) are preferably expressed in the display library. The substrate and the peptide may be linked via the N- or C-terminus of the peptide or substrate as further described below.

The compounds of the present invention may be made by standard synthetic methods, recombinant DNA techniques, or any other method of producing peptides and fusion proteins. Compounds of the present invention that include non-peptide moieties may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions, if applicable.

A primary intended use for the compounds of the invention is as therapeutic or prophylactic agents. In this context, it is a particular advantage of the invention that the peptide coupled to the support may have an activity comparable to - or even higher than, the natural ligand mimicked (mimetic) by the peptide. An additional advantage is that, furthermore, certain natural, ligand-based, therapeutic reagents can induce antibodies against the patient's own endogenous antibodies; The substrate bound peptides avoid this trap due to their typical sequence identity with the natural ligand, or only a small part of the sequence identity.

The compounds of the present invention - while intended in accordance with the present invention for use as therapeutic or prophylactic agents, may also be used to screen for such agents. For example, an Fc-peptide (for example, the Fc-peptide domain of SH2) may be used in an anti-Fc coated plate assay. The support, especially the Fc, can make the insoluble peptides soluble and therefore useful in a variety of assays.

The compounds of the present invention can be used for therapeutic or prophylactic purposes after formulating them with suitable pharmaceutical carrier materials by administering an effective amount to a patient, such as a human or other mammal, in need thereof.

A number of further advantages of the present invention will become apparent from consideration of the accompanying drawings and the following detailed description of the invention.

Brief description of the drawings

Fig. 1 shows schematically an exemplary method according to the invention. In this preferred method, the support is an Fc domain, covalently linked to the peptide by expression from a DNA construct encoding both the Fc domain and the peptide. As indicated in Figure 1, the Fc domains spontaneously form a dimer in this method.

Fig. 2 shows exemplary Fc dimers that can be derived from an IgG1 antibody. "Fc" in Figure 1 represents any Fc variant encompassed by the term "Fc domain" as used herein. "X¹"And" X²"Represent peptides or linker-peptide combinations as defined below. Particular dimers are:

A, D: Single disulfide bonded dimers. IgG1 antibodies typically have two disulfide bonds in the region of junction between the constant and variable domains. The Fc domain shown in Figures 2A and 2D may be formed by cleavage between the sites of two disulfide bonds or by substituting a cysteinyl residue with a non-reactive (for example, an alanyl) residue. In Fig. 2A, the Fc domain is fused to the amino terminus of the peptides; in Fig. 2D, to the carboxyl ends.

B, E: Dimers doubly bonded with a disulfide bond. This Fc domain can be created by truncation of the original antibody to retain both cysteinyl residues in the Fc domain chains, or by expression of a construct comprising a sequence encoding such an Fc domain. In Fig. 2B, the Fc domain is fused to the amino terminus of the peptides; in Fig. 2E - to the carboxyl ends.

C, F: Non-covalent dimers. This Fc domain can be generated by elimination of a cysteinyl residue either by truncation or by substitution. Elimination of the cysteinyl residue may be desirable to avoid contamination that may arise from the reaction of the cysteinyl residue with cysteinyl residues of other proteins present in the host cell. Non-covalent binding of the Fc domains is sufficient to keep the dimer intact. Other dimers can be made using Fc domains derived from different types of antibodies (for example,

IgG2, IgM).

Figure 3 shows the structure of preferred compounds of the invention having tandem repeats of a pharmacologically active peptide. Fig. 3A shows a single chain molecule and may also show a DNA construct for that molecule. Fig. 3B shows a dimer where the linker-peptide system is present in only one dimer chain. Fig. 3C shows a dimer having a peptide portion in both chains. The dimer shown in Fig. 3C will be generated spontaneously in certain host cells upon expression of a single-chain DNA construct shown in Figure 3A. In the case of other host cells, the cells could be placed under conditions that favor dimer formation, or dimers could be obtained in vitro.

Fig. 4 shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of the human IgG1 Fc domain that may be used in the present invention.

Fig. 5 shows a synthetic scheme for the preparation of PEGylated peptide 19 (SEQ ID NO: 3).

Fig. 6 shows a synthetic scheme for the preparation of pegylated peptide 20 (SEQ ID NO: 4).

Figure 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, below.

Figure 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, below.

Figure 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, below.

Figure 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, below.

Figure 11 shows the amount of plaques generated in vivo in normal female BDF1 mice administered by single injection with a bolus dose of 100 µg / kg of various compounds, where the terms are defined as follows:

PEG-MGDF is an average molecular weight PEG attached to the N-terminal amino group by reductive amination of amino acids 1-163 of native human TPO expressed in E. coli (so that it is not glycosylated)

TMP: a TPO peptide mimetic having the amino acid sequence of IEGPTLRQWLAARA (SEQ ID NO: 13);

TMP-TMP: a TPO peptide mimetic having the amino acid sequence IEGPTLRQWLAARAGGGGGGGG-IEGPTLRQWLAARA (SEQ ID NO: 14);

PEG-TMP-TMP: a peptide having the sequence of SEQ ID NO: 14, wherein the PEG group has an average molecular weight of 5 kD, PEG attached as shown in Figure 6;

Fc-TMP-TMP: compound of SEQ ID NO: 8 (Fig. 8) dimerized with a second identical monomer (i.e., Cys residues at positions 7 and 10 are linked to corresponding Cys residues of the second monomer to form a dimer, as shown in Fig. 2); while

TMP-TMP-Fc is a compound with the sequence of SEQ ID NO: 10 (Fig. 9) dimerized in the same way as TMP-TMP-Fc except that the Fc domain is attached to the C-terminus of the TMP-TMP peptide rather than to the N the end of this peptide.

Fig. 12 shows the amount of platelets generated in vivo in normal BDF1 mice dosed with the various compounds via implantable osmotic pumps over a period of 7 days. These compounds are defined in the same manner as above with reference to Figure 7.

Figure 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, below.

Figure 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, below.

Figure 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, below.

Figure 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, below.

PL 211 164 B1

Figures 17A and 17B show the DNA sequence (SEQ ID NO: 23) inserted into pCFM1656 between the unique AatII restriction sites (position # 4364 in pCFM1656) and SacII (position # 4585 in pCFM1656) to generate the expression plasmid pAMG21 (ATCC access No. 98113 ).

Fig. 18A illustrates the in vivo generated hemoglobin, red blood cells and hematocrit levels in normal female BDF1 mice administered by a single bolus injection of 100 µg / kg of various compounds. Fig. 18B shows the same results in mice dosed at 100 µg / kg / day by a 7-day micro-osmotic pump with different EMPs at 100 µg / kg, rhEPO at 30U / mouse. (In both experiments, neutrophils, lymphocytes and platelets were not influenced). In these figures, the terms are defined as follows:

Fc-EMP: compound with the sequence SEQ ID NO: 16 (Fig. 13) dimerized with a second identical monomer (i.e., Cys residues at positions 7 and 10 are linked to corresponding Cys residues of the second monomer to form a dimer as shown in Fig. 2) ; while

EMP-Fc: is a compound with the sequence of SEQ ID NO: 18 (Fig. 14) dimerized in the same manner as Fc-EMP except that the Fc domain is attached to the C-terminus of the EMP peptide rather than to the N-terminus of the peptide.

"EMP-EMP-Fc" refers to a tandem repeat of the same peptide (SEQ ID NO: 20) joined to the same Fc domain by the carboxyl terminus of the peptides. "Fc-EMP-EMP" refers to the same tandem peptide repeat, with the same Fc domain attached to the amino terminus of the tandem repeat. All molecules are expressed in E. coli and are therefore not glycosylated.

Figures 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, below.

Figures 20A and 20B show the nucleotide and amino acid sequences (SEQ ID NOS: 1057 and 1058) of the TNF-u-Fc inhibitor fusion molecule described in Example 4, below.

Figures 21A and 21B show the nucleotide and amino acid sequences (SEQ ID NOS: 1059 and 1060) of the Fc-11L-1 antagonist fusion molecule described in Example 5, below.

Figures 22A and 22B show the nucleotide and amino acid sequences (SEQ ID NOS: 1061 and 1062) of the IL-1-Fc antagonist fusion molecule described in Example 5, below.

Figures 23A, 23B, and 23C show the nucleotide and amino acid sequences (SEQ ID NOS: 1063 and 1064) of the Fc-antagonist VEGF fusion molecule described in Example 6, below.

Figures 24A and 24B show the nucleotide and amino acid sequences (SEQ ID NOS: 1065 and 1066) of the VEGF-Fc antagonist fusion molecule described in Example 6, below.

Figures 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, below.

Figures 26A and 26B show the nucleotide and amino acid sequences (SEQ ID NOS: 1069 and 1070) of the MMP-Fc inhibitor fusion molecule described in Example 7, below.

Detailed Description of the Invention Definitions of Terms

Terms used in this specification are defined as follows unless limited in particular instances.

The term "comprising" means that the compound may include additional amino acids at one or both of the N- or C- ends of the sequence in question. Of course, these additional amino acids should not significantly affect the activity of the compound.

The term "substrate" refers to a molecule that prevents degradation and / or increases half-life, lowers toxicity, lowers immunogenicity, or increases the biological activity of a therapeutic protein. Exemplary substrates include: an Fc domain (which is preferred) as well as a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); branched-chain polymer (see for example, U.S. Patent No. 4,289,872 to Denkenwalter et al., issued September 15, 1981; 5,229,490 to: Tam, issued Jul 20, 1993; WO. 93/21259 by Frechet et al., Issued Oct. 28, 1993); lipid; a cholesterol group (such as a steroid); hydrocarbon or oligosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a "salvage" receptor. The substrates are described further below.

The term "native Fc" refers to a molecule or sequence comprising the sequence of the non-antigen-binding fragment resulting from the digestion of a whole antibody, in monomeric form.

PL 211 164 B1 or multimeric. The original native Fc immunoglobulin source is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's are composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent linkage. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from 1 to 4 depending on the class (e.g., TgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). An example of a native Fc is a disulfide-linked dimer obtained from digestion of IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term "native Fc" as used in this application is a genus term for monomeric, dimeric and multimeric forms.

The term "variant Fc" refers to a molecule or sequence that is a modified native Fc but still includes the binding site of the "salvage" receptor, FcRn. International applications WO 97/34631 (published September 25, 1997) and WO 96/32478 describe exemplary Fc variants as well as interaction with the "salvage" receptor and are incorporated herein by reference. Thus, the term "Fc variant" includes a molecule or sequence that is "humanized" from a non-human native Fc. Furthermore, native Fc includes sites that can be removed as they provide structural features or biological activity that are not required for the fusion molecule of the present invention. Thus, the term "Fc variant" includes a molecule or sequence that lacks one or more native Fc sites or residues that influence or are associated with (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than the "salvage" receptor; or (7) antibody dependent cellular cytotoxicity (ADCC). Fc variants are described in more detail below.

The term "Fc domain" includes native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc variants, the term "Fc domain" includes molecules in monomeric or multimeric form, digested from whole antibodies, or otherwise made.

The term "multimeter" when referring to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains joined covalently, non-covalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM - pentamers; IgD - dimers; IgA - monomers, dimers, trimers or tetramers. Multimers can be formed by utilizing the sequence and resulting activity of the native Ig Fc source, or by derivatizing (as defined below) such native Fc.

The term "dimer" as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains, covalently or non-covalently linked. Thus, exemplary dimers within the scope of the present invention are as set out in Fig. 2.

The terms "derivatization" and "derivative" or "derivatized" include processes and compounds resulting therefrom, respectively wherein (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues in a compound; (2) the compound is cross-linked or cross-linking is taking place; for example, the compound has a cysteinyl residue and thereby forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl bonds is replaced by a non-peptidyl bond: (4) the N-terminus is therefore replaced by -NRR¹, NRC (O) R¹, -NRC (O) OR¹, -NRS (O)₂R¹, -NHC- (O) NHR, succinimide, or substituted or unsubstituted benzyloxycarbonyl-NH-, where R and R¹ ring substituents are as defined below; (5) The C-terminus is replaced

3 4 2 3 4 by -C (O) R² or -NR³R⁴where R.², R³ and r⁴ are as defined below and (6) compounds in which particular amino acid moieties are modified by treatment with agents capable of reacting with selected side chains or terminal residues. The derivatives are further described below.

The term "peptide" refers to molecules of 2 to 40 amino acids, with 3 to 20 amino acids being preferred and 6 to 15 amino acids being the most preferred. Exemplary peptides may be randomly generated by any of the methods cited above, performed in a peptide library (e.g., a phage display library), or derived by protein digestion.

The term "randomized" as used in reference to a peptide sequence refers to fully randomized sequences (for example, selected by phage display methods) and sequences where

One or more residues of the naturally occurring molecule are replaced by an amino acid residue not appearing at that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, RNA-peptide screening, chemical screening, etc.

The term "pharmacologically active" means that the substance so described has an 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 include agonist or mimetic and antagonist peptides as defined below.

The terms "-mimetic peptide" and "agonistic peptide" refer to a peptide with a biological activity comparable to a protein (for example EPO, TPO, G-CSF) which interacts with the protein in question. The terms further include peptides which indirectly mimic the activity of the protein concerned, such as by enhancing the effect of the natural ligand of the protein concerned; see for example, G-CSF-mimetic peptides listed in Tables 2 and 7. Thus, the term "EPO-mimetic peptide" includes any peptides that can be identified or derived as described in Wrighton et al. (1996), 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 containing an EPO-mimetic object. Those of ordinary skill in the art will appreciate that each link allows the selection of peptides other than those actually disclosed in this application by the following disclosed procedures with different peptide libraries.

The term "TPO-mimetic peptide" includes peptides that can be identified or derived as described in Cwirla et al. (1997), Science 276: 1696-9, US Pat. 5,869,451 and 5,932,946 and any other reference in Table 2 identified as pertaining to a TPOmimetic object as well as in the US patent application, "Thrombopoietic Compounds," filed on the same date as this application and incorporated herein by reference. Those of ordinary skill in the art will appreciate that each of these links enables the selection of peptides other than those actually disclosed in this application by the following disclosed procedures with different peptide libraries.

The term "G-CSF-mimetic peptide" includes any peptides that can be identified or described in: Paukovits et al. (1984), Hoppe-Seylers Z., Physiol. Chem. 365: 303-11 or any of the references in Table 2 identified as containing a G-CSF mimetic object. Conventional skilled artisans will appreciate that each of these links enables them to select peptides other than those actually disclosed in this application by the following disclosed procedures with different peptide libraries.

The term "CTLA4-mimetic peptide" includes any peptides that can be identified or derived as described in Fukumoto et al. (1998), Nature Biotech., 16: 267-70. It will be appreciated by those skilled in the art that each of these links enables them to select peptides other than those actually disclosed in this application by the following disclosed procedures with different peptide libraries.

The term "antagonist peptide" or "inhibitor peptide" refers to a peptide which blocks or in some way interferes with the biological activity of the bound protein of interest, or has a biological activity comparable to a known antagonist or inhibitor of the considered bound protein. Thus, the term "TNF-antagonist peptide" includes peptides that can be identified or derived as described in Takasaki et al. (1997), Nature Biotech. 15: 1266-70 or any of the references in Table 2 identified as containing a TNF-antagonist target. It will be appreciated by those skilled in the art that each of these links enables them to select peptides other than those actually disclosed in this application by the following disclosed procedures with different peptide libraries.

The terms "IL-1 antagonist" and "IL-1ra-mimetic peptide" include peptides that inhibit or down-regulate IL-1 receptor activation by IL-1. Activation of the Il-1 receptor results from the formation of a complex between Il-1, the Il-1 receptor and the helper protein of the IL-1 receptor. IL-1 antagonist or IL-1ra-mimetic peptides bind to IL-1, IL-1 receptor or IL-1 receptor helper protein and inhibit complex formation between any two or three members of the complex. Exemplary IL-1 antagonist or IL-1ra mimetic peptides may be identified or derived as described in U.S. Patent Nos. 5,608,035, 5,786,331, 5,880,096, or any of the references in Table 2 identified as containing an IL-1a-mimetic or IL-1 antagonist object. . It will be appreciated by those ordinarily skilled in the art that each of these links enables them to select different peptides than those actually disclosed in this application by the following disclosed procedures with different peptide libraries.

The term "VFGF-antagonist peptide" includes peptides that can be identified or derived as described in Fairbrother (1998), Biochem. 37: 17754-54, and in any of the references in Table 2 identified as containing a VEGF-antagonist object. It will be appreciated by those ordinarily skilled in the art that each of these links enables them to select other peptides than those actually disclosed in this application by the following disclosed procedures with different peptide libraries.

The term "peptide MMP inhibitor" includes peptides that can be identified or derived as described in Koivunen (1999). Nature Biotech. 17: 768-74 and in any of the references in Table 2 identified as containing an MMP inhibitory object. It will be appreciated by those of ordinary skill in the art that each of these links enables them to select other peptides than actually disclosed in this application by the following disclosed procedures with different peptide libraries.

In addition, physiologically acceptable salts of the compounds of the present invention are also included in this application. By "physiologically acceptable salts" is meant any salts that are known or later discovered to be pharmaceutically acceptable. Some specific examples are: acetate, trifluoroacetate, hydrochlorides such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate and oxalate.

The structure of relationships.

Generally. In the compositions of the substances produced according to the present invention, the peptide may be attached to the support via the N-terminus or the C-terminus of the peptide. Thus, the substrate-peptide molecules of the present invention can be described by the following formula I:

I (X¹) aF¹- (X²) b where:

F.¹ is a substrate (preferably an Fc domain);

X¹ and X² are each independently selected from: - (L.¹) cP¹, - (L.¹) cP¹- (L.²) dP², - (L.¹) cP¹- (L.²) dP²- (L.³) eP³, and - (L.¹) cP¹- (L.²) dP²- (L.³) eP³- (L.⁴) fP⁴

P.¹, P², P³, and P⁴ represent each independently the sequences of pharmacologically active peptides;

L.¹, L.², L.³, and L.⁴ denote 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.

Thus, compound I includes the preferred compounds of formula

II X¹-F¹ and their multimers, where F¹ is an Fc domain and is attached to the C-terminus of the X¹;

III F¹-X² and their multimers, in which F.¹ is an Fc domain and is attached at the N-terminus of the X²;

IV F¹- (L.¹) cP¹ and their multimers, in which F.¹ is an Fc domain and is attached at the N-terminus (L¹) cP¹; and

VF¹- (L.¹) cP¹- (L.²) dP² and their multimers, where F¹ is an Fc domain and is attached at the N-terminus -L¹-P¹-L²-P².

Peptides. Any number of peptides may be used in conjunction with the present invention. Of particular interest are peptides mimicking 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, especially those antagonizing the activity of TNF, leptin, any of the interleukins (IL-1, 2, 3, ...) and proteins associated with the activation of complement (e.g., C3b). Targeting peptides are also of interest, including tumor targeting peptides, membrane transport peptides, etc. All of these classes of peptides can be discovered by methods described in the references cited in this specification and other references.

Phage display, in particular, is useful in the generation of peptides for use in the present invention. It has been found that affinity selection from random peptide libraries can be used to identify ligands for any site of any gene product. Dedman et al. (1993), J. Biol. Chem. 268: 23025-30. Phage display is particularly well suited to identifying peptides that bind to such proteins of concern as cell surface receptors or any proteins with linear epitopes. Wilson Mn .. (1998), Can.

PL 211 164 B1

J. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins are widely described in Herz et al. (1997), J. Receptor &amp; amp; Signal Transduction Res. 17 (5): 671-776 which is incorporated herein by reference to this text. Such contemplated proteins are preferred for use in the present invention.

A particularly preferred group of peptides are those which bind to cytokine receptors. Cytokines have recently been classified according to their receptor code. See Inglot (1997), Archivum Immunologiae et Terapiae Experimentalis 45: 353-7, which is hereby incorporated by reference into this text. Of these receptors, the most preferred are: CKR (family I in Table 3). The classification of the receptors appears in Table 3.

Table 3 - Cytokine receptors classified by the receptor code

<td colspan="2">Cytokines (ligands)</td><td colspan="4">Receptor type</td>

<td>Family</td><td>Under the family</td><td colspan="2">family</td><td colspan="2">subfamily</td>

<td>I. Cytokines</td><td>1. IL-2, IL-4, IL-7, IL-9, IL-13, IL-15</td><td colspan="2">I. Cytokine R (CKR)</td><td> 1.</td><td>shared YCr</td>

<td>Hematopoietic</td><td>2. IL-3, IL-5, GM-CSF</td><td></td><td></td><td> 2.</td><td>shared GP 140 ER</td>

<td></td><td>3. IL-6, IL-11, IL-12, LIF, OSM,</td><td></td><td></td><td> 3.</td><td>3. Common RP 130</td>

<td></td><td>CNTF, Leptin (OB) 4. G-CSF, EPO, TPO, PRL, GH</td><td></td><td></td><td> 4.</td><td>"One string" R.</td>

<td></td><td>5. IL-17, HVS-IL-17</td><td></td><td></td><td> 5.</td><td>another R.^c</td>

<td>II. IL-10 ligands</td><td>IL-10, BCRF-1, HSV-IL-10</td><td>II.</td><td>IL-10 R</td><td></td><td></td>

<td>III. Interferons</td><td>1. IFN-a1, α2, α4, m, t, IFN-e^d2. IFN-γ</td><td>III.</td><td>Interferon R.</td><td> 1. 2.</td><td>IFNAR IFNGR</td>

<td>IV. IL-1 ligands</td><td>1. IL-1a, IL-1e, IL-1Ra</td><td>IV</td><td>IL-1R</td><td></td><td></td>

<td>V. TNF ligands</td><td>1. TNF-α, TNF-β (LT), FAS1, CD40 L, CD30 L, CD27 L</td><td>V.</td><td>NGF / TNF R^e</td><td></td><td></td>

<td></td><td>1.α chemokines: IL-8, GRO α, β, γ,</td><td></td><td></td><td> 1.</td><td>CXCR</td>

<td></td><td>IF-10, PF-4, SDF-1</td><td></td><td></td><td></td><td></td>

<td>VI. Chemokines</td><td>2. β chemokines: MlP1a, MIP1 β,</td><td></td><td></td><td></td><td></td>

<td></td><td>MCP-1,2,3,4, RANTES, eotaxin</td><td>VI.</td><td>Chemokine R.</td><td> 2.</td><td>CCR</td>

<td></td><td>3. γ chemokines: lymphotactin</td><td></td><td></td><td> 3.</td><td>CR</td>

<td></td><td></td><td></td><td></td><td> 4.</td><td>DARC^f</td>

<td>VII. Growth factors</td><td>1.1 SCF, M-CSF, PDGF-AA, AB,</td><td>VI</td><td>. RKF</td><td> 1.</td><td>TK sub-family</td>

<td></td><td>BB, FLT-3L, VEGF, SSV-PDGF</td><td></td><td></td><td> 1.</td><td>1 IgTK III R</td>

<td></td><td>1.2 FGFa, FGFe</td><td></td><td></td><td> 1.</td><td>2 IgTK IV R</td>

<td></td><td>1.3 EGF, TGF-a, VV-F19 (similar</td><td></td><td></td><td> 1.</td><td>3 TK-I rich in Cysteine</td>

<td></td><td>do-EGF)</td><td></td><td></td><td></td><td></td>

<td></td><td>1.4 IGF-I, IGF-II, Insulin</td><td></td><td></td><td> 1.</td><td>4 TK-II rich in Cysteine</td>

<td></td><td>1.5 NGF, BDNF, NT-3, NT-4^g</td><td></td><td></td><td> 1.</td><td>5 Cysteine ​​node TKV</td>

<td></td><td>2. TGF-e1, e2, e3</td><td></td><td></td><td> 2.</td><td>sub-family of STK^h</td>

^c IL-17R belongs to the CKR family but is not assigned to any of the 4 indicated subfamilies.

^d Other type I IFN subtypes remain unassigned. Hematopoietic cytokines, IL-10 ligands, and interferons lack internal functional protein kinases. The signaling molecules for cytokines are JAK molecules, STAT molecules, and other related non-receptor molecules. IL-14, IL-16 and IL-18 have been cloned but remain unassigned according to the receptor code.

^e TNF receptors use multiple, distinct intracellular molecules for signal transduction, including "FAS R dead dementia and 55 kDa TNF-aR," which are involved in their cytotoxic effects. NGF / TNF R can bind both NGF and related factors as well as TNF ligands. Chemokine receptors are coupled to the G protein, seven transmembrane domain receptors (7TM, serpentine).

^f DARC - Duffie's blood group antigen is an erythrocyte receptor that has the ability to bind several different chemokines. Belong to the immunoglobulin superfamily, but the characteristics of its signal transduction events remain unclear.

^g Neurotrophic cytokines can also bind to NGF / TNF receptors.

^h The STKS can include many other TGF β related factors that remain unassigned. Protein kinases are an integral part of the intracellular domain of the kinase family receptor (RKF). Enzymes are involved in signal transmission through receptors.

PL 211 164 B1

Exemplary peptides for the present invention include Tables 4 to 20 below. These peptides can be made by methods disclosed in the art. Single letter abbreviations of amino acid names are used. The symbol "X" in these sequences (and throughout this specification - unless otherwise stated in a specific instance) means that any of the 20 naturally occurring amino acid residues may be present. Any of these peptides can be linked tandem (that is, sequentially) with or without a linker, and several examples of tandem linked are provided in this table. Linkers are marked with a "Λ" and may be any linkers described herein. Tandem repeats and linkers are shown separated by dashes for clarity. Each peptide containing a cysteine ​​residue may be cross-linked with another peptide containing Cys, either or both of which may be attached to a support. A few cross-linked examples are given in this table. Any peptide having more than one Cys residue can also form an intra-peptide disulfide bond; see, for example, EPO-mimetic peptides in Table 5. Several examples of peptides with an intra-peptide disulfide bond are given in this table. Each of these peptides can be derivatized as described herein, and several derivatized examples are given in the table. The derivatized peptides in the table below are exemplary rather than limiting as the respective non-derivatized peptides may be used in the present invention. For derivatives in which the carboxy terminus is closed with an amino group, the ending amino group is designated -NH2. For derivatives in which the amino acid residues are substituted with a moiety other than amino acid residues, these substitutions are indicated by the symbol σ, which means any residue described in Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9 and Cuthbertson et al. (1997), J. Med. Chem. 40: 2875-82, which are cited by way of reference. The J substituents and the Z substituents (Z₅, WITH₆, ... WITH₄₀) are defined in U.S. Patent Nos. 5,608,035, 5,786,331, and 5,880,096, which are incorporated herein by reference. For EPOmimetic sequences (Table 5), the substituents X2 to X11 and the integer "n" are as defined in WO 96/40772, which is incorporated herein by reference. The substituents "Ψ", "Θ" and "+" are as defined in Sparks et al. (1996), Proc. Natl. Acad. Sci. 93: 1540-4, which is incorporated herein by reference. X4, X5, X6 and X7 are as defined in U.S. Patent No. 5,773,569, which is hereby incorporated by reference, except that the integrin binding peptides X1, X2, X3, X4, X5, X6, X7 and X8 are as defined in the international application WO 95/14714, published June 1, 1995 and WO 97/08203, published March 6, 1997, which are also incorporated by reference, and for the 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 October 30, 1997, also incorporated by reference. Xaa and Yaa below are as defined in WO 98/09985, published March 12, 1998, incorporated by reference. AA1, AA2, AB1, AB2 and AC are as defined in WO 98/53842, published December 3, 1998, incorporated by reference. X¹, X², X³ and X⁴ only in Table 17 are they as defined in European application EP 0 911 393, published April 28, 1999. Remains in bold are D-amino acids. All peptides are linked by peptide bonds unless otherwise stated. The abbreviations are listed at the end of this description. In the column headed "SEQ ID NO", the symbol "NR" means that it is not necessary to show sequences in the sequence listing.

Table 4 - Sequences of the peptide IL-1 antagonists

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>Z11 Z7Z8QZ₅YZ6ZgZ10</td><td> 212</td>

<td>XXQZ₅YZ6XX</td><td> 907</td>

<td>Z7XQZ₅YZ6XX</td><td> 908</td>

<td>Z7Z8QZ₅YZ6ZgZ10</td><td> 909</td>

<td>Z11 Z7Z8QZ₅YZ6ZgZ10</td><td> 910</td>

<td>Z12Z13Z14Z15Z16Z17Z18Z1gZ20Z21 Z22Z11Z7Z8QZ5YZ6Z9Z10L</td><td> 917</td>

<td>Z23NZ24Z3gZ25Z26Z27Z28Z2gZ30Z40</td><td> 979</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>TANVSSFEWTPYYWQPYALPL</td><td> 213</td>

<td>SWTDYGYWQPYALPISGL</td><td> 214</td>

<td>ETPFTWEESNAYYWQPYALPL</td><td> 215</td>

<td>ENTYSPNWADSMYWQPYALPL</td><td> 216</td>

<td>SVGEDHNFWTSEYWQPYALPL</td><td> 217</td>

<td>DGYDRWRQSGERYWQPYALPL</td><td> 218</td>

<td>FEWTPGYWQPY</td><td> 219</td>

<td>FEWTPGYWQHY</td><td> 220</td>

<td>FEWTPGWYQJY</td><td> 221</td>

<td>AcFEWTPGWYQJY</td><td> 222</td>

<td>FEWTPGWpYQJY</td><td> 223</td>

<td>FAWTPGYWQJY</td><td> 224</td>

<td>FEWAPGYWQJY</td><td> 225</td>

<td>FEWVPGYWQJY</td><td> 226</td>

<td>FEWTPGYWQJY</td><td> 227</td>

<td>AcFEWTPGYWQJY</td><td> 228</td>

<td>FEWTPaWYQJY</td><td> 229</td>

<td>FEWTPSarWYQJY</td><td> 230</td>

<td>FEWTPGYYQPY</td><td> 231</td>

<td>FEWTPGWWQPY</td><td> 232</td>

<td>FEWTPNYWQPY</td><td> 233</td>

<td>FEWTPvYWQJY</td><td> 234</td>

<td>FEWTPecGYWQJY</td><td> 235</td>

<td>FEWTPAibYWQJY</td><td> 236</td>

<td>FEWTSarGYWQJY</td><td> 237</td>

<td>FEWTPGYWQPY</td><td> 238</td>

<td>FEWTPGYWQHY</td><td> 239</td>

<td>FEWTPGWYQJY</td><td> 240</td>

<td>AcFEWTPGWYQJY</td><td> 241</td>

<td>FEWTPGW-pY-QJY</td><td> 242</td>

<td>FAWTPGYWQJY</td><td> 243</td>

<td>FEWAPGYWQJY</td><td> 244</td>

<td>FEWVPGYWQJY</td><td> 245</td>

<td>FEWTPGYWQJY</td><td> 246</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>AcFEWTPGYWQJY</td><td> 247</td>

<td>FEWTPAWYQJY</td><td> 248</td>

<td>FEWTPSarWYQJY</td><td> 249</td>

<td>FEWTPGYYQPY</td><td> 250</td>

<td>FEWTPGWWQPY</td><td> 251</td>

<td>FEWTPNYWQPY</td><td> 252</td>

<td>FEWTPVYWQJY</td><td> 253</td>

<td>FEWTPecGYWQJY</td><td> 254</td>

<td>FEWTPAibYWQJY</td><td> 255</td>

<td>FEWTSarGYWQJY</td><td> 256</td>

<td>FEWTPGYWQPYALPL</td><td> 257</td>

<td>1 NapEWTPGYYQJY</td><td> 258</td>

<td>YEWTPGYYQJY</td><td> 259</td>

<td>FEWVPGYYQJY</td><td> 260</td>

<td>FEWTPSYYQJY</td><td> 261</td>

<td>FEWTPNYYQJY</td><td> 262</td>

<td>TKPR</td><td> 263</td>

<td>RKSSK</td><td> 264</td>

<td>RKQDK</td><td> 265</td>

<td>NRKQDK</td><td> 266</td>

<td>RKQDKR</td><td> 267</td>

<td>ENRKQDKRF</td><td> 268</td>

<td>VTKFYF</td><td> 269</td>

<td>VTKFY</td><td> 270</td>

<td>VTDFY</td><td> 271</td>

<td>SHLYWQPYSVQ</td><td> 671</td>

<td>TLVYWQPYSLQT</td><td> 672</td>

<td>RGDYWQPYSVQS</td><td> 673</td>

<td>VHVYWQPYSVQT</td><td> 674</td>

<td>RLVYWQPYSVQT</td><td> 675</td>

<td>SRVWFQPYSLQS</td><td> 676</td>

<td>NMVYWQPYSIQT</td><td> 677</td>

<td>SVVFWQPYSVQT</td><td> 678</td>

<td>TFVYWQPYALPL</td><td> 679</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>TLVYWQPYSIQR</td><td> 680</td>

<td>RLVYWQPYSVQR</td><td> 681</td>

<td>SPVFWQPYSIQI</td><td> 682</td>

<td>WIEWWQPYSVQS</td><td> 683</td>

<td>SLIYWQPYSLQM</td><td> 684</td>

<td>TRLYWQPYSVQR</td><td> 685</td>

<td>RCDYWQPYSVQT</td><td> 686</td>

<td>MRVFWQPYSVQN</td><td> 687</td>

<td>KIVYWQPYSVQT</td><td> 688</td>

<td>RHLYWQPYSVQR</td><td> 689</td>

<td>ALVWWQPYSEQI</td><td> 690</td>

<td>SRVWFQPYSLQS</td><td> 691</td>

<td>WEQPYALPLE</td><td> 692</td>

<td>QLVWWQPYSVQR</td><td> 693</td>

<td>DLRYWQPYSVQV</td><td> 694</td>

<td>ELVWWQPYSLQL</td><td> 695</td>

<td>DLVWWQPYSVQW</td><td> 696</td>

<td>NGNYWQPYSFQV</td><td> 697</td>

<td>ELVYWQPYSIQR</td><td> 698</td>

<td>ELMYWQPYSVQE</td><td> 699</td>

<td>NLLYWQPYSMQD</td><td> 700</td>

<td>GYEWYQPYSVQR</td><td> 701</td>

<td>SRVWYQPYSVQR</td><td> 702</td>

<td>LSEQYQPYSVQR</td><td> 703</td>

<td>GGGWWQPYSVQR</td><td> 704</td>

<td>VGRWYQPYSVQR</td><td> 705</td>

<td>VHVYWQPYSVQR</td><td> 706</td>

<td>QARWYQPYSVQR</td><td> 707</td>

<td>VHVYWQPYSVQT</td><td> 708</td>

<td>RSVYWQPYSVQR</td><td> 709</td>

<td>TRVWFQPYSVQR</td><td> 710</td>

<td>GRIWFQPYSVQR</td><td> 711</td>

<td>GRVWFQPYSVQR</td><td> 712</td>

<td>ARTWYQPYSVQR</td><td> 713</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>ARVWWQPYSVQM</td><td> 714</td>

<td>RLMFYQPYSVQR</td><td> 715</td>

<td>ESMWYQPYSVQR</td><td> 716</td>

<td>HFGWWQPYSVHM</td><td> 717</td>

<td>ARFWWQPYSVQR</td><td> 718</td>

<td>RLVYWQ PYAPIY</td><td> 719</td>

<td>RLVYWQ PYSYQT</td><td> 720</td>

<td>RLVYWQ PYSLPI</td><td> 721</td>

<td>RLVYWQ PYSVQA</td><td> 722</td>

<td>SRVWYQ PYAKGL</td><td> 723</td>

<td>SRVWYQ PYAQGL</td><td> 724</td>

<td>SRVWYQ PYAMPL</td><td> 725</td>

<td>SRVWYQ PYSVQA</td><td> 726</td>

<td>SRVWYQ PYSLGL</td><td> 727</td>

<td>SRVWYQ PYAREL</td><td> 728</td>

<td>SRVWYQ PYSRQP</td><td> 729</td>

<td>SRVWYQ PYFVQP</td><td> 730</td>

<td>EYEWYQ PYALPL</td><td> 731</td>

<td>IPEYWQ PYALPL</td><td> 732</td>

<td>SRIWWQ PYALPL</td><td> 733</td>

<td>DPLFWQ PYALPL</td><td> 734</td>

<td>SRQWVQ PYALPL</td><td> 735</td>

<td>IRSWWQ PYALPL</td><td> 736</td>

<td>RGYWQ PYALPL</td><td> 737</td>

<td>RLLWVQ PYALPL</td><td> 738</td>

<td>EYRWFQ PYALPL</td><td> 739</td>

<td>DAYWVQ PYALPL</td><td> 740</td>

<td>WSGYFQ PYALPL</td><td> 741</td>

<td>NIEFWQ PYALPL</td><td> 742</td>

<td>TRDWVQ PYALPL</td><td> 743</td>

<td>DSSWYQ PYALPL</td><td> 744</td>

<td>IGNWYQ PYALPL</td><td> 745</td>

<td>NLRWDQ PYALPL</td><td> 746</td>

<td>LPEFWQ PYALPL</td><td> 747</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>DSYWWQ PYALPL</td><td> 748</td>

<td>RSQYYQ PYALPL</td><td> 749</td>

<td>ARFWLQ PYALPL</td><td> 750</td>

<td>NSYFWQ PYALPL</td><td> 751</td>

<td>RFMYWQPYSVQR</td><td> 752</td>

<td>AHLFWQPYSVQR</td><td> 753</td>

<td>WWQPYALPL</td><td> 754</td>

<td>YYQPYALPL</td><td> 755</td>

<td>YFQPYALGL</td><td> 756</td>

<td>YWYQPYALPL</td><td> 757</td>

<td>RWWQPYATPL</td><td> 758</td>

<td>GWYQPYALGF</td><td> 759</td>

<td>YWYQPYALGL</td><td> 760</td>

<td>IWYQPYAMPL</td><td> 761</td>

<td>SNMQPYQRLS</td><td> 762</td>

<td>TFVYWQPY AVGLPAAETACN</td><td> 763</td>

<td>TFVYWQPY SVQMTITGKVTM</td><td> 764</td>

<td>TFVYWQPY SSHXXVPXGFPL</td><td> 765</td>

<td>TFVYWQPY YGNPQWAIHVRH</td><td> 766</td>

<td>TFVYWQPY VLLELPEGAVRA</td><td> 767</td>

<td>TFVYWQPY VDYVWPIPIAQV</td><td> 768</td>

<td>GWYQPYVDGWR</td><td> 769</td>

<td>RWEQPYVKDGWS</td><td> 770</td>

<td>EWYQPYALGWAR</td><td> 771</td>

<td>GWWQPYARGL</td><td> 772</td>

<td>LFEQPYAKALGL</td><td> 773</td>

<td>GWEQPYARGLAG</td><td> 774</td>

<td>AWVQPYATPLDE</td><td> 775</td>

<td>MWYQPYSSQPAE</td><td> 776</td>

<td>GWTQPYSQQGEV</td><td> 777</td>

<td>DWFQPYSIQSDE</td><td> 778</td>

<td>PWIQPYARGFG</td><td> 779</td>

<td>RPLYWQPYSVQV</td><td> 780</td>

<td>TLIYWQPYSVQI</td><td> 781</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>RFDYWQPYSDQT</td><td> 782</td>

<td>WHQFVQPYALPL</td><td> 783</td>

<td>EWDS VYWQPYSVQ TLLR</td><td> 784</td>

<td>WEQN VYWQPYSVQ SFAD</td><td> 785</td>

<td>SDV VYWQPYSVQ SLEM</td><td> 786</td>

<td>YYDG VYWQPYSVQ VMPA</td><td> 787</td>

<td>SDIWYQ PYALPL</td><td> 788</td>

<td>QRIWWQ PYALPL</td><td> 789</td>

<td>SRIWWQ PYALPL</td><td> 790</td>

<td>RSLYWQ PYALPL</td><td> 791</td>

<td>TIIWEQ PYALPL</td><td> 792</td>

<td>WETWYQ PYALPL</td><td> 793</td>

<td>SYDWEQ PYALPL</td><td> 794</td>

<td>SRIWCQ PYALPL</td><td> 795</td>

<td>EIMFWQ PYALPL</td><td> 796</td>

<td>DYVWQQ PYALPL</td><td> 797</td>

<td>MDLLVQ WYQPYALPL</td><td> 798</td>

<td>GSKVIL WYQPYALPL</td><td> 799</td>

<td>RQGANI WYQPYALPL</td><td> 800</td>

<td>GGGDEP WYQPYALPL</td><td> 801</td>

<td>SQLERT WYQPYALPL</td><td> 802</td>

<td>ETWVRE WYQPYALPL</td><td> 803</td>

<td>KKGSTQ WYQPYALPL</td><td> 804</td>

<td>LQARMN WYQPYALPL</td><td> 805</td>

<td>EPRSQKWYQPYALPL</td><td> 806</td>

<td>VKQKWR WYQPYALPL</td><td> 807</td>

<td>LRRHDV WYQPYALPL</td><td> 808</td>

<td>RSTASI WYQPYALPL</td><td> 809</td>

<td>ESKEDQ WYQPYALPL</td><td> 810</td>

<td>EGLTMK WYQPYALPL</td><td> 811</td>

<td>EGSREG WYQPYALPL</td><td> 812</td>

<td>VIEWWQ PYALPL</td><td> 813</td>

<td>VWYWEQ PYALPL</td><td> 814</td>

<td>ASEWWQ PYALPL</td><td> 815</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>FYEWWQ PYALPL</td><td> 816</td>

<td>EGWWVQ PYALPL</td><td> 817</td>

<td>WGEWLQ PYALPL</td><td> 818</td>

<td>DYVWEQ PYALPL</td><td> 819</td>

<td>AHTWWQ PYALPL</td><td> 820</td>

<td>FIEWFQ PYALPL</td><td> 821</td>

<td>WLAWEQ PYALPL</td><td> 822</td>

<td>VMEWWQ PYALPL</td><td> 823</td>

<td>ERMWQ PYALPL</td><td> 824</td>

<td>NXXWXX PYALPL</td><td> 825</td>

<td>WGNWYQ PYALPL</td><td> 826</td>

<td>TLYWEQ PYALPL</td><td> 827</td>

<td>VWRWEQ PYALPL</td><td> 828</td>

<td>LLWTQ PYALPL</td><td> 829</td>

<td>SRIWXX PYALPL</td><td> 830</td>

<td>SDIWYQ PYALPL</td><td> 831</td>

<td>WGYYXX PYALPL</td><td> 832</td>

<td>TSGWYQ PYALPL</td><td> 833</td>

<td>VHPYXX PYALPL</td><td> 834</td>

<td>EHSYFQ PYALPL</td><td> 835</td>

<td>XXIWYQ PYALPL</td><td> 836</td>

<td>AQLHSQ PYALPL</td><td> 837</td>

<td>WANWFQ PYALPL</td><td> 838</td>

<td>SRLYSQ PYALPL</td><td> 839</td>

<td>GVTFSQ PYALPL</td><td> 840</td>

<td>SIVWSQ PYALPL</td><td> 841</td>

<td>SRDLVQ PYALPL</td><td> 842</td>

<td>HWGH VYWQPYSVQ DDLG</td><td> 843</td>

<td>SWHS VYWQPYSVQ SVPE</td><td> 844</td>

<td>WRDS VYWQPYSVQ PESA</td><td> 845</td>

<td>TWDA VYWQPYSVQ KWLD</td><td> 846</td>

<td>TPPW VYWQPYSVQ SLDP</td><td> 847</td>

<td>YWSS VYWQPYSVQ SVHS</td><td> 848</td>

<td>YWY QPY ALGL</td><td> 849</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>YWY QPY ALPL</td><td> 850</td>

<td>EWI QPY ATGL</td><td> 851</td>

<td>NWE QPY AKPL</td><td> 852</td>

<td>AFY QPY ALPL</td><td> 853</td>

<td>FLY QPY ALPL</td><td> 854</td>

<td>VCK QPY LEWC</td><td> 855</td>

<td>ETPFTWEESNAYYWQPYALPL</td><td> 856</td>

<td>QGWLTWQDSVDMYWQPYALPL</td><td> 857</td>

<td>FSEAGYTWPENTYWQPYALPL</td><td> 858</td>

<td>TESPGGLDWAKIYWQPYALPL</td><td> 859</td>

<td>DGYDRWRQSGERYWQPYALPL</td><td> 860</td>

<td>TANVSSFEWTPGYWQPYALPL</td><td> 861</td>

<td>SVGEDHNFWTSE YWQPYALPL</td><td> 862</td>

<td>MNDQTSEVSTFP YWQPYALPL</td><td> 863</td>

<td>SWSEAFEQPRNL YWQPYALPL</td><td> 864</td>

<td>QYAEPSALNDWG YWQPYALPL</td><td> 865</td>

<td>NGDWATADWSNY YWQPYALPL</td><td> 866</td>

<td>THDEHI YWQPYALPL</td><td> 867</td>

<td>MLEKTYTTWTPG YWQPYALPL</td><td> 868</td>

<td>WSDPLTRDADL YWQPYALPL</td><td> 869</td>

<td>SDAFTTQDSQAM YWQPYALPL</td><td> 870</td>

<td>GDDAAWRTDSLT YWQPYALPL</td><td> 871</td>

<td>AIIRQLYRWSEM YWQPYALPL</td><td> 872</td>

<td>ENTYSPNWADSM YWQPYALPL</td><td> 873</td>

<td>MNDQTSEVSTFP YWQPYALPL</td><td> 874</td>

<td>SVGEDHNFWTSE YWQPYALPL</td><td> 875</td>

<td>QTPFTWEESNAY YWQPYALPL</td><td> 876</td>

<td>ENPFTWQESNAY YWQPYALPL</td><td> 877</td>

<td>VTPFTWEDSNVF YWQPYALPL</td><td> 878</td>

<td>QIPFTWEQSNAY YWQPYALPL</td><td> 879</td>

<td>QAPLTWQESAAY YWQPYALPL</td><td> 880</td>

<td>EPTFTWEESKAT YWQPYALPL</td><td> 881</td>

<td>TTTLTWEESNAY YWQPYALPL</td><td> 882</td>

<td>ESPLTWEESSAL YWQPYALPL</td><td> 883</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>ETPLTWEESNAY YWQPYALPL</td><td> 884</td>

<td>EATFTWAESNAY YWQPYALPL</td><td> 885</td>

<td>EALFTWKESTAY YWQPYALPL</td><td> 886</td>

<td>STP-TWEESNAY YWQPYALPL</td><td> 887</td>

<td>ETPFTWEESNAY YWQPYALPL</td><td> 888</td>

<td>KAPFTWEESQAY YWQPYALPL</td><td> 889</td>

<td>STSFTWEESNAY YWQPYALPL</td><td> 890</td>

<td>DSTFTWEESNAY YWQPYALPL</td><td> 891</td>

<td>YIPFTWEESNAY YWQPYALPL</td><td> 892</td>

<td>QTAFTWEESNAY YWQPYALPL</td><td> 893</td>

<td>ETLFTWEESNAT YWQPYALPL</td><td> 894</td>

<td>VSSFTWEESNAY YWQPYALPL</td><td> 895</td>

<td>QPYALPL</td><td> 896</td>

<td>Py-1-NapPYQJYALPL</td><td> 897</td>

<td>TANVSSFEWTPG YWQPYALPL</td><td> 898</td>

<td>FEWTPGYWQPYALPL</td><td> 899</td>

<td>FEWTPGYWQJYALPL</td><td> 900</td>

<td>FEWTPGYYQJYALPL</td><td> 901</td>

<td>ETPFTWEESNAYYWQPYALPL</td><td> 902</td>

<td>FTWEESNAYYWQJYALPL</td><td> 903</td>

<td>ADVL YWQPYA PVTLWV</td><td> 904</td>

<td>GDVAE YWQPYA LPLTSL</td><td> 905</td>

<td>SWTDYG YWQPYA LPISGL</td><td> 906</td>

<td>FEWTPGYWQPYALPL</td><td> 911</td>

<td>FEWTPGYWQJYALPL</td><td> 912</td>

<td>FEWTPGWYQPYALPL</td><td> 913</td>

<td>FEWTPGWYQJYALPL</td><td> 914</td>

<td>FEWTPGYYQPYALPL</td><td> 915</td>

<td>FEWTPGYYQJYALPL</td><td> 916</td>

<td>TANVSSFEWTPGYWQPYALPL</td><td> 918</td>

<td>SWTDYGYWQPYALPISGL</td><td> 919</td>

<td>ETPFTWEESNAYYWQPYALPL</td><td> 920</td>

<td>ENTYSPNWADSMYWQPYALPL</td><td> 921</td>

<td>SVGEDHNFWTSEYWQPYALPL</td><td> 922</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>DGYDRWRQSGERYWQPYALPL</td><td> 923</td>

<td>FEWTPGYWQPYALPL</td><td> 924</td>

<td>FEWTPGYWQPY</td><td> 925</td>

<td>FEWTPGYWQJY</td><td> 926</td>

<td>EWTPGYWQPY</td><td> 927</td>

<td>FEWTPGWYQJY</td><td> 928</td>

<td>AEWTPGYWQJY</td><td> 929</td>

<td>FAWTPGYWQJY</td><td> 930</td>

<td>FEATPGYWQJY</td><td> 931</td>

<td>FEWAPGYWQJY</td><td> 932</td>

<td>FEWTAGYWQJY</td><td> 933</td>

<td>FEWTPAYWQJY</td><td> 934</td>

<td>FEWTPGAWQJY</td><td> 935</td>

<td>FEWTPGYAQJY</td><td> 936</td>

<td>FEWTPGYWQJA</td><td> 937</td>

<td>FEWTGGYWQJY</td><td> 938</td>

<td>FEWTPGYWQJY</td><td> 939</td>

<td>FEWTJGYWQJY</td><td> 940</td>

<td>FEWTPecGYWQJY</td><td> 941</td>

<td>FEWTPAibYWQJY</td><td> 942</td>

<td>FEWTPSarWYQJY</td><td> 943</td>

<td>FEWTSarGYWQJY</td><td> 944</td>

<td>FEWTPNYWQJY</td><td> 945</td>

<td>FEWTPVYWQJY</td><td> 946</td>

<td>FEWTVPYWQJY</td><td> 947</td>

<td>AcFEWTPGWYQJY</td><td> 948</td>

<td>AcFEWTPGYWQJY</td><td> 949</td>

<td>1 Nap-EWTPGYYQJY</td><td> 950</td>

<td>YEWTPGYYQJY</td><td> 951</td>

<td>FEWVPGYYQJY</td><td> 952</td>

<td>FEWTPGYYQJY</td><td> 953</td>

<td>FEWTPsYYQJY</td><td> 954</td>

<td>FEWTPNYYCJY</td><td> 955</td>

<td>SHLY-Nap-QPYSVQM</td><td> 956</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>TLVY-Nap-QPYSLQT</td><td> 957</td>

<td>RGDY-Nap-QPYSVQS</td><td> 958</td>

<td>NMVY-Nap-QPYSIQT</td><td> 959</td>

<td>VYWQPYSVQ</td><td> 960</td>

<td>VY-Nap-QPYSVQ</td><td> 961</td>

<td>TFVYWQJYALPL</td><td> 962</td>

<td>FEWTPGYYQJ-Bpa</td><td> 963</td>

<td>XaaFEWTPGYYQJ-Bpa</td><td> 964</td>

<td>FEWTPGY-Bpa-QJY</td><td> 965</td>

<td>AcFEWTPGY-Bpa-QJY</td><td> 966</td>

<td>FEWTPG-Bpa-YQJY</td><td> 967</td>

<td>AcFEWTPG-Bpa-YQJY</td><td> 968</td>

<td>AcFE-Bpa-TPGYYQJY</td><td> 969</td>

<td>AcFE-Bpa-TPGYYQJY</td><td> 970</td>

<td>Bpa-EWTPGYYQJY</td><td> 971</td>

<td>AcBpa-EWTPGYYQJY</td><td> 972</td>

<td>VYWQPYSVQ</td><td> 973</td>

<td>RLVYWQPYSVQR</td><td> 974</td>

<td>RLVY-Nap-QPYSVQR</td><td> 975</td>

<td>RLDYWQPYSVQR</td><td> 976</td>

<td>RLVWFQPYSVQR</td><td> 977</td>

<td>RLVYWQPYSIQR</td><td> 978</td>

<td>DNSSWYDSFLL</td><td> 980</td>

<td>DNTAWYESFLA</td><td> 981</td>

<td>DNTAWYENFLL</td><td> 982</td>

<td>PARE DNTAWYDSFLI WC</td><td> 983</td>

<td>TSEY DNTTWYEKFLA SQ</td><td> 984</td>

<td>SQIP DNTAWYQSFLL HG</td><td> 985</td>

<td>SPFI DNTAWYENFLL YOU</td><td> 986</td>

<td>EQIY DNTAWYDHFLL SY</td><td> 987</td>

<td>TPFI DNTAWYENFLL YOU</td><td> 988</td>

<td>TITES DNTAWYERFLM SY</td><td> 989</td>

<td>TMTQ DNTAWYENFLL SY</td><td> 990</td>

<td>TI DNTAWYANLVQ TYPQ</td><td> 991</td>

PL 211 164 B1 cont. table 4

<td> 1</td><td> 2</td>

<td>TI DNTAWYERFLA QYPD</td><td> 992</td>

<td>HI DNTAWYENFLL TYTP</td><td> 993</td>

<td>SQ DNTAWYENFLL SYKA</td><td> 994</td>

<td>QI DNTAWYERFLL QYNA</td><td> 995</td>

<td>NQ DNTAWYESFLL QYNT</td><td> 996</td>

<td>TI DNTAWYENFLL NHNL</td><td> 997</td>

<td>HY DNTAWYERFLQ QGWH</td><td> 998</td>

<td>ETPFTWEESNAYYWQPYALPL</td><td> 999</td>

<td>YIPFTWEESNAYYWQPYALPL</td><td> 1000</td>

<td>DGYDRWRQSGERYWQPYALPL</td><td> 1001</td>

<td>pY-1 Nap-pY-QJYALPL</td><td> 1002</td>

<td>TANVSSFEWTPGYWQPYALPL</td><td> 1003</td>

<td>FEWTPGYWQJYALPL</td><td> 1004</td>

<td>FEWTPGYWQPYALPLSD</td><td> 1005</td>

<td>FEWTPGYYQJYALPL</td><td> 1006</td>

<td>FEWTPGYWQJY</td><td> 1007</td>

<td>AcFEWTPGYWQJY</td><td> 1008</td>

<td>AcFEWTPGWYQJY</td><td> 1009</td>

<td>AcFEWTPGYYQJY</td><td> 1010</td>

<td>AcFEWTPaYWQJY</td><td> 1011</td>

<td>AcFEWTPaWYQJY</td><td> 1012</td>

<td>AcFEWTPaYYQJY</td><td> 1013</td>

<td>FEWTPGYYQJYALPL</td><td> 1014</td>

<td>FEWTPGYWQJYALPL</td><td> 1015</td>

<td>FEWTPGWYQJYALPL</td><td> 1016</td>

<td>TANVSSFEWTPGYWQPYALPL</td><td> 1017</td>

<td>AcFEWTPGYWQJY</td><td> 1018</td>

<td>AcFEWTPGWYQJY</td><td> 1019</td>

<td>AcFEWTPGYYQJY</td><td> 1020</td>

<td>AcFEWTPAYWQJY</td><td> 1021</td>

<td>AcFEWTPAWYQJY</td><td> 1022</td>

<td>AcFEWTPAYYQJY</td><td> 1023</td>

PL 211 164 B1

Table 5 - Sequences of EPO-mimetic peptides

<td>Sequence / structure</td><td>SEQ ID NO:</td>

<td>YXCXXGPXTWXCXP</td><td> 83</td>

<td>YXCXXGPXTWXCXP-YXCXXGPXTWXCXP</td><td> 84</td>

<td>YXCXXGPXTWXCXP-A-YXCXXGPXTWXCXP</td><td> 85</td>

<td>YXCXXGPXTWXCXP-A- _with . _x</td><td> 86</td>

<td>\ ^ (s-amine)</td><td></td>

<td>K. /</td><td></td>

<td>/ βΑ YXCXXGPXTWXCXP-A-^WITH (a-amine)</td><td> 86</td>

<td>GGTYSCHFGPLTWVCKPQGG</td><td> 87</td>

<td>GGDYHCRMGPLTWYCKPLGG</td><td> 88</td>

<td>GGVYACRMGPITWVCSPLGG</td><td> 89</td>

<td>VGNYMCHFGPITWVCRPGGG</td><td> 90</td>

<td>GGLYLCRFGPVTWDCGYKGG</td><td> 91</td>

<td>GGTYSCHFGPLTWVCKPQGG- GGTYSCHFGPLTWVCKPQGG</td><td> 92</td>

<td>GGTYSCHFGPLTWVCKPQGG -ΛGGTYSCHFGPLTWVCKPQGG</td><td> 93</td>

<td>GGTYSCHFGPLTWVCKPQGGSSK</td><td> 94</td>

<td>GGTYSCHFGPLTWVCKPQGGSSK- GGTYSCHFGPLTWVCKPQGGSSK</td><td> 95</td>

<td>GGTYSCHFGPLTWVCKPQGGSSK-A- GGTYSCHFGPLTWVCKPQGGSSK</td><td> 96</td>

<td>GGTYSCHFGPLTWVCKPQGGSS. \ (ε-amine)</td><td> 97 '</td>

<td>K. / /^βΑGGTYSCHFGPLTWVCKPQGGSS (a-amine)</td><td> 97</td>

<td>GGTYSCHFGPLTWVCKPQGGSSK (-A-biotin)</td><td> 98</td>

<td>CX₄X₅GPX₆TWX₇C.</td><td> 421</td>

<td>GGTYSCHGPLTWVCKPQGG</td><td> 422</td>

<td>VGNYMAHMGPITWVCRPGG</td><td> 423</td>

<td>GGPHHYYACRMGPLTWIC</td><td> 424</td>

<td>GGTYSCHFGPLTWVCKPQ</td><td> 425</td>

<td>GGLYACHMGPMTWVCQPLRG</td><td> 426</td>

<td>TIAQYICYMGPETWECRPSPKA</td><td> 427</td>

<td>YSCHFGPLTWYCK</td><td> 428</td>

<td>YCHFGPLTWVC</td><td> 429</td>

<td>X₃X4X5GPX₆TWX₇X8</td><td> 124</td>

<td>YX2X₃X4X5GPX₆TWX₇X8</td><td> 461</td>

<td>XiYX₂X3X4X5GPX₆TWX₇X8X₉XioXi i</td><td> 419</td>

<td>XiYX₂CX4X5GPX₆TWX₇CX₉XioXi i</td><td> 420</td>

<td>GGLYLCRFGPVTWDCGYKGG</td><td> 1024</td>

<td>GGTYSCHFGPLTWVCKPQGG</td><td> 1025</td>

<td>GGDYHCRMGPLTWYCKPLGG</td><td> 1026</td>

<td>VGNYMCHFGPITWVCRPGGG</td><td> 1029</td>

<td>GGVYACRMGPITWVCSPLGG</td><td> 1030</td>

<td>VGNYMAHMGPITWVCRPGG</td><td> 1035</td>

<td>GGTYSCHFGPLTWVCKPQ</td><td> 1036</td>

<td>GGLYACHMGPMTWVCQPLRG</td><td> 1037</td>

<td>TIAQYICYMGPETWECRPSPKA</td><td> 1038</td>

<td>YSCHFGPLTWVCK</td><td> 1039</td>

<td>YCHFGPLTWYC</td><td> 1040</td>

<td>SCHFGPLTWYCK</td><td> 1041</td>

<td>(AX₂) nX3X4XsGPX₆TWX₇X₈</td><td> 1042</td>

PL 211 164 B1

Table 6 - Sequences of TPO-mimetic peptides

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>IEGPTLRQWLAARA</td><td> 13</td>

<td>IEGPTLRQWLAAKA</td><td> 24</td>

<td>LEGPTLREWLAAR</td><td> 25</td>

<td>IEGPTLRQWLAARA-A-IEGPTLRQWLAARA</td><td> 26</td>

<td>IEGPTLRQWLAAKA-A-IEGPTLRQWLAAKA</td><td> 27</td>

<td>IEGPTLRQCLAARA-A-IEGPTLRQCLAARA II</td><td> 28</td>

<td>IEGPTLRQWLAARA-AK (BrAc) -A- - IEGPTLRQWLAARA</td><td> 29</td>

<td>IEGPTLRQWLAARA-AK (PEG) - Λ-IEGPTLRQWLAARA</td><td> 30</td>

<td>IEGPTLRQCLAARA-A-IEGPTLRQWLAARA</td><td> 31</td>

<td>1 IEGPTLRQCLAARA-A-IEGPTLRQWLAARA</td><td> 31</td>

<td>IEGPTLRQWLAARA-A-IEGPTLRQCLAARA</td><td> 32</td>

<td>1 IEGPTLRQWLAARA-A-IEGPTLRQCLAARA</td><td> 32</td>

<td>VRDQIXXXL</td><td> 33</td>

<td>TLREWL</td><td> 34</td>

<td>GRVRDQVAGW</td><td> 35</td>

<td>GRVKDQIAQL</td><td> 36</td>

<td>GVRDQVSWAL</td><td> 37</td>

<td>ESVREQVMKY</td><td> 38</td>

<td>SVRSQISASL</td><td> 39</td>

<td>GVRETVYRHM</td><td> 40</td>

<td>GVREVIVMHML</td><td> 41</td>

<td>GRVRDQIWAAL</td><td> 42</td>

<td>AGVRDQILIWL</td><td> 43</td>

<td>GRVRDQIMLSL</td><td> 44</td>

<td>GRVRDQI (X) aL</td><td> 45</td>

<td>CTLRQWLQGC</td><td> 46</td>

<td>CTLQEFLEGC</td><td> 47</td>

<td>CTRTEWLHGC</td><td> 48</td>

<td>CTLREWLHGGFC</td><td> 49</td>

<td>CTLREWVFAGLC</td><td> 50</td>

<td>CTLRQWLILLGMC</td><td> 51</td>

<td>CTLAEFLASGVEQC</td><td> 52</td>

PL 211 164 B1 cont. table 6

<td> 1</td><td> 2</td>

<td>CSLQEFLSHGGYVC</td><td> 53</td>

<td>CTLREFLDPTTAVC</td><td> 54</td>

<td>CTLKEWLVSHEVWC</td><td> 55</td>

<td>CTLREWL (X) 2-6C</td><td> 56-60</td>

<td>REGPTLRQWM</td><td> 61</td>

<td>EGPTLRQWLA</td><td> 62</td>

<td>ERGPFWAKAC</td><td> 63</td>

<td>REGPRCVMWM</td><td> 64</td>

<td>CGTEGPTLSTWLDC</td><td> 65</td>

<td>CEQDGPTLLEWLKC</td><td> 66</td>

<td>CELVGPSLMSWLTC</td><td> 67</td>

<td>CLTGPFVTQWLYEC</td><td> 68</td>

<td>CRAGPTLLEWLTLC</td><td> 69</td>

<td>CADGPTLREWISFC</td><td> 70</td>

<td>C (X) 1-2EGPTLREWL (X) 1-2C</td><td> 71-74</td>

<td>GGCTLREWLHGGFCGG</td><td> 75</td>

<td>GGCADGPTLREWISFCGG</td><td> 76</td>

<td>GNADGPTLRQWLEGRRPKN</td><td> 77</td>

<td>LAIEGPTLRQWLHGNGRDT</td><td> 78</td>

<td>HGRVGPTLREWKTQVATKK</td><td> 79</td>

<td>TIKGPTLRQWLKSREHTS</td><td> 80</td>

<td>ISDGPTLKEWLSVTRGAS</td><td> 81</td>

<td>SIEGPTLREWLTSRTPHS</td><td> 82</td>

Table 7 - G-CSF-mimetic peptide sequences

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>EEDCK</td><td> 99</td>

<td>EEDCK</td><td> 99</td>

<td>| EEDCK</td><td> 99</td>

<td>EEDσK</td><td> 100</td>

<td>EEDσK</td><td> 100</td>

<td>| EEDσK</td><td> 100</td>

<td>pGgluEDσK</td><td> 101</td>

<td>pGluEDσK</td><td> 101</td>

<td>| pGluEDσK</td><td> 101</td>

PL 211 164 B1 cont. table 7

<td> 1</td><td> 2</td>

<td>PicSDσK</td><td> 102</td>

<td>PicSDσK</td><td> 102</td>

<td>| PicSDσK</td><td> 102</td>

<td>EEDCK-A-EEDCK</td><td> 103</td>

<td>EEDXK-A-EEDXK</td><td> 104</td>

Table 8 - Sequences of peptide TNF antagonists

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td>YCFTASENHCY</td><td> 106</td>

<td>YCFTNSENHCY</td><td> 107</td>

<td>YCFTRSENHCY</td><td> 108</td>

<td>FCASENHCY</td><td> 109</td>

<td>YCASENHCY</td><td> 110</td>

<td>FCNSENHCY</td><td> 111</td>

<td>FCNSENRCY</td><td> 112</td>

<td>FCNSVENRCY</td><td> 113</td>

<td>YCSQSVSNDCF</td><td> 114</td>

<td>FCVSNDRCY</td><td> 115</td>

<td>YCRKELGQVCY</td><td> 116</td>

<td>YCKEPGQCY</td><td> 117</td>

<td>YCRKEMGCY</td><td> 118</td>

<td>FCRKEMGCY</td><td> 119</td>

<td>YCWSQNLCY</td><td> 120</td>

<td>YCELSQYLCY</td><td> 121</td>

<td>YCWSQNYCY</td><td> 122</td>

<td>YCWSQYLCY</td><td> 123</td>

<td>DFLPHYKNTSLGHRP</td><td> 1085</td>

<td>AA1-AB1 \ AC</td><td>NO</td>

<td>AA2AB2 /</td><td></td>

PL 211 164 B1

Table 9 - Integrin binding peptide sequences

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>RX1ETX2WX3</td><td> 441</td>

<td>RX1ETX2WX3</td><td> 442</td>

<td>RGDGX</td><td> 443</td>

<td>CRGDGXC</td><td> 444</td>

<td>CX1X2RLDX₃X4C</td><td> 445</td>

<td>CARRLDAPC</td><td> 446</td>

<td>CPSRLDSPC</td><td> 447</td>

<td>X1X2XaRGDX4X₅X6</td><td> 448</td>

<td>CX2CRGDCX₅C.</td><td> 449</td>

<td>CDCRGDCFC</td><td> 450</td>

<td>CDCRGDCLC</td><td> 451</td>

<td>CLCRGDCIC</td><td> 452</td>

<td>X1X2DDX4X₅X? X8</td><td> 453</td>

<td>X1X2X3DDX4X₅X₅X7X8</td><td> 454</td>

<td>CWDDGWLC</td><td> 455</td>

<td>CWDDLWWLC</td><td> 456</td>

<td>CWDDGLMC</td><td> 457</td>

<td>CWDDGWMC</td><td> 458</td>

<td>CSWDDGWLC</td><td> 459</td>

<td>CPDDLWWLC</td><td> 460</td>

<td>NGR</td><td>NO</td>

<td>GSL</td><td>NO</td>

<td>RGO</td><td>NO</td>

<td>CGRECPRLCQSSC</td><td> 1071</td>

<td>CNGRCVSGCAGRC</td><td> 1072</td>

<td>CLSGSLSC</td><td> 1073</td>

<td>RGD</td><td>NO</td>

<td>NGR</td><td>NO</td>

<td>GSL</td><td>NO</td>

<td>NGRAHA</td><td> 1074</td>

<td>CNGRC</td><td> 1075</td>

<td>COCRGDCFC</td><td> 1076</td>

<td>CGSLVRC</td><td> 1077</td>

PL 211 164 B1 cont. table 9

<td> 1</td><td> 2</td>

<td>DLXXL</td><td> 1043</td>

<td>RTDLDSLRTYTL</td><td> 1044</td>

<td>RTDLDSLRTY</td><td> 1053</td>

<td>RTDLDSLRT</td><td> 1054</td>

<td>RTDLOSLR</td><td> 1078</td>

<td>GDLDLLKLRLTL</td><td> 1079</td>

<td>GDLHSLRQLLSR</td><td> 1080</td>

<td>RDDLHMLRLQLW</td><td> 1081</td>

<td>SSDLHALKKRYG</td><td> 1082</td>

<td>RGDLKQLSELTW</td><td> 1083</td>

<td>RGDLAALSAPPV</td><td> 1084</td>

Table 10 - Sequences of the peptide antagonists of Selectin

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>DITWDQLWDLMK</td><td> 147</td>

<td>DITWDELWKIMN</td><td> 148</td>

<td>DYTWFELWDMMQ</td><td> 149</td>

<td>QITWAQLWNMMK</td><td> 150</td>

<td>DMTWHDLWTLMS</td><td> 151</td>

<td>DYSWHDLWEMMS</td><td> 152</td>

<td>EITWDQLWEVMN</td><td> 153</td>

<td>HVSWEQLWDIMN</td><td> 154</td>

<td>HITWDQLWRIMT</td><td> 155</td>

<td>RNMSWLELWEHMK</td><td> 156</td>

<td>AEWTWDQLWHVMNPAESQ</td><td> 157</td>

<td>HRAEWLALWEQMSP</td><td> 158</td>

<td>KKEDWLALWRIMSV</td><td> 159</td>

<td>ITWDQLWDLMK</td><td> 160</td>

<td>DITWDQLWDLMK</td><td> 161</td>

<td>DITWDQLWDLMK</td><td> 162</td>

<td>DITWDQLWDLMK</td><td> 163</td>

<td>CQNRYTDLVAIQNKNE</td><td> 462</td>

<td>AENWADNEPNNKRNNED</td><td> 463</td>

<td>RKNNKTWTWVGTKKALTNE</td><td> 464</td>

PL 211 164 B1 cont. table 10

<td> 1</td><td> 2</td>

<td>KKALTNEAENWAD</td><td> 465</td>

<td>CQXRYTDLVAIQNKXE</td><td> 466</td>

<td>RKXNXXWTWVGTXKXLTEE</td><td> 467</td>

<td>AENWADGEPNNKXNXED</td><td> 468</td>

<td>CXXXYTXLVAIQNKXE</td><td> 469</td>

<td>RKXXXXWXWVGTXKXLTXE</td><td> 470</td>

<td>AXNWXXXEPNNXXXED</td><td> 471</td>

<td>XKXKTXEAXNWXX</td><td> 472</td>

Table 11 - Sequences of anti-pathogenic peptides

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>GFFALIPKIISSPLFKTLLSAVGSALSSSGGQQ</td><td> 503</td>

<td>GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE</td><td> 504</td>

<td>GFFALIPKIISSPLFKTLLSAV</td><td> 505</td>

<td>GFFALIPKIISSPLFKTLLSAV</td><td> 506</td>

<td>KGFFALIPKIISSPLFKTLLSAV</td><td> 507</td>

<td>KKGFFALIPKIISSPLFKTLLSAV</td><td> 508</td>

<td>KKGFFALIPKIISSPLFKTLLSAV</td><td> 509</td>

<td>GFFALIPKIIS</td><td> 510</td>

<td>GIGAVLKVLTTGLPALISWIKRKRQQ</td><td> 511</td>

<td>GIGAVLKVLTTGLPALISWIKRKRQQ</td><td> 512</td>

<td>GIGAVLKVLTTGLPALISWIKRKRQQ</td><td> 513</td>

<td>GIGAVLKVLTTGLPALISWIKR</td><td> 514</td>

<td>AVLKVLTTGLPALISWIKR</td><td> 515</td>

<td>KLLLLLKLLLLK</td><td> 516</td>

<td>KLLLKLLLKLLK</td><td> 517</td>

<td>KLLLKLKLKLLK</td><td> 518</td>

<td>KKLLKLKLKLKK</td><td> 519</td>

<td>KLLLKLLLKLLK</td><td> 520</td>

<td>KLLLKLKLKLLK</td><td> 521</td>

<td>KLLLLK</td><td> 522</td>

<td>KLLLKLLK</td><td> 523</td>

PL 211 164 B1 cont. table 11

<td> 1</td><td> 2</td>

<td>KLLLKLKLKLLK</td><td> 524</td>

<td>KLLLKLKLKLLK</td><td> 525</td>

<td>KLLLKLKLKLLK</td><td> 526</td>

<td>KAAAKAAAKAAK</td><td> 527</td>

<td>KVVVKVVVKVVK</td><td> 528</td>

<td>KVVVKVKVKVVK</td><td> 529</td>

<td>KVVVKVKVKVK</td><td> 530</td>

<td>KVVVKVKVKVVK</td><td> 531</td>

<td>KLILKL</td><td> 532</td>

<td>KVLHLL</td><td> 533</td>

<td>LKLRLL</td><td> 534</td>

<td>KPLHLL</td><td> 535</td>

<td>KLILKLYR</td><td> 536</td>

<td>KVFHLLHL</td><td> 537</td>

<td>HKFRILKL</td><td> 538</td>

<td>KPFHILHL</td><td> 539</td>

<td>KIIIKIKIIK</td><td> 540</td>

<td>KIIIKIKIIK</td><td> 541</td>

<td>KIIIKIKIIK</td><td> 542</td>

<td>KIPIKIKIKIPK</td><td> 543</td>

<td>KIPIKIKIKIVK</td><td> 544</td>

<td>RIIIRIRIRIIR</td><td> 545</td>

<td>RIIIRIRIRIIR</td><td> 546</td>

<td>RIIIRIRIRIIR</td><td> 547</td>

<td>RIVIRIRIRLIR</td><td> 548</td>

<td>RIIVRIRLRIIR</td><td> 549</td>

<td>RIGIRLRVRIIR</td><td> 550</td>

<td>KIVIRIRIRLIR</td><td> 551</td>

<td>RIAVKWRLRFIK</td><td> 552</td>

<td>KIGWKLRVRIIR</td><td> 553</td>

<td>KKIGWLIIRVRR</td><td> 554</td>

<td>RIVIRIRIRLIRIR</td><td> 555</td>

PL 211 164 B1 cont. table 11

<td> 1</td><td> 2</td>

<td>RIIVRIRLRIIRVR</td><td> 556</td>

<td>RIGIRLRVRIIRRV</td><td> 557</td>

<td>KIVIRIRARLIRIRIR</td><td> 558</td>

<td>RIIVKIRLRIIKKIRL</td><td> 559</td>

<td>KIGIKARVRIIRVKII</td><td> 560</td>

<td>RI IVHIRLRIIHHIRL</td><td> 561</td>

<td>HIGIKAHVRIIRVHII</td><td> 562</td>

<td>RIYVKIHLRYIKKIRL</td><td> 563</td>

<td>KIGHKARVHIIRYKII</td><td> 564</td>

<td>RIYVKPHPRYIKKIRL</td><td> 565</td>

<td>KPGHKARPHIIRYKII</td><td> 566</td>

<td>KIVIRIRIRLIRIRIRKIV</td><td> 567</td>

<td>RIIVKIRLRIIKKIRLIKK</td><td> 568</td>

<td>KIGWKLRVRIIRVKIGRLR</td><td> 569</td>

<td>KIVIRIRIRLIRIRIRKIVKVKRIR</td><td> 570</td>

<td>RFAVKIRLRIIKKIRLIKKIRKRVIK</td><td> 571</td>

<td>KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK</td><td> 572</td>

<td>RIYVKPHPRYIKKIRL</td><td> 573</td>

<td>KPGHKARPHIIRYKII</td><td> 574</td>

<td>KIVIRIRIRLIRIRIRKIV</td><td> 575</td>

<td>RIIVKIRLRIIKKIRLIKK</td><td> 576</td>

<td>RIYVSKISIYIKKIRL</td><td> 577</td>

<td>KIVIFTRIRLTSIRIRSIV</td><td> 578</td>

<td>KPIHKARPTIIRYKMI</td><td> 579</td>

<td>Cyclical CKGFFALIPKIISSPLFKTLLSAVC</td><td> 580</td>

<td>CKKGFFALIPKIISSPLFKTLLSAVC</td><td> 581</td>

<td>CKKKGFFALIPKIISSPLFKTLLSAVC</td><td> 582</td>

<td>Cyclic CRIVIRIRIRLIRIRC</td><td> 583</td>

<td>Cyclic CKPGHKARPHIIRYKIIC</td><td> 584</td>

<td>Cyclic CRFAVKIRLRIIKKIRLIKKIRKRVIKC</td><td> 585</td>

<td>KLLLKLLL KLLKC</td><td> 586</td>

<td>KLLLKLLLKLLK</td><td> 587</td>

<td>KLLLKLKLKLLKC</td><td> 588</td>

<td>KLLLKLLLKLLK</td><td> 589</td>

PL 211 164 B1

Table 12 - Sequences of VlP-mimetic peptides

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>HSDAVFYDNYTR LRKQMAVKKYLN MOT</td><td> 590</td>

<td>Nle HSDAVFYDNYTR LRKQMAVKKYLN SILN</td><td> 591</td>

<td>X ·, X1 Χ1 Χ2</td><td> 592</td>

<td>X3 S X4 LN</td><td> 593</td>

<td>NH CH CO KKYX5 NH CH CO X6 | | (CH2) m Z (CH2) n</td><td> 594</td>

<td>KKYL</td><td> 595</td>

<td>NSILN</td><td> 596</td>

<td>KKYL</td><td> 597</td>

<td>KKYA</td><td> 598</td>

<td>AVKKYL</td><td> 599</td>

<td>NSILN</td><td> 600</td>

<td>KKYV</td><td> 601</td>

<td>SILauN</td><td> 602</td>

<td>KKYLNle</td><td> 603</td>

<td>NSYLN</td><td> 604</td>

<td>NSIYN</td><td> 605</td>

<td>KKYLPPNSILN</td><td> 606</td>

<td>LauKKYL</td><td> 607</td>

<td>CapKKYL</td><td> 608</td>

<td>KYL</td><td>NO</td>

<td>KKYNle</td><td> 609</td>

<td>VKKYL</td><td> 610</td>

<td>LNSILN</td><td> 611</td>

<td>YLNSILN</td><td> 612</td>

<td>KKYLN</td><td> 613</td>

<td>KKYLNS</td><td> 614</td>

<td>KKYLNSI</td><td> 615</td>

<td>KKYLNSIL</td><td> 616</td>

<td>KKYL</td><td> 617</td>

PL 211 164 B1 cont. table 12

<td></td><td> 1</td><td> 2</td>

<td>KKYDA</td><td></td><td> 618</td>

<td>AVKKYL</td><td></td><td> 619</td>

<td>NSILN</td><td></td><td> 620</td>

<td>KKYV</td><td></td><td> 621</td>

<td>SILauN</td><td></td><td> 622</td>

<td>NSYLN</td><td></td><td> 623</td>

<td>NSIYN</td><td></td><td> 624</td>

<td>KKYLNle</td><td></td><td> 625</td>

<td>KKYLPPNSILN</td><td></td><td> 626</td>

<td>KKYL</td><td></td><td> 627</td>

<td>KKYDA</td><td></td><td> 628</td>

<td>AVKKYL</td><td></td><td> 629</td>

<td>NSILN</td><td></td><td> 630</td>

<td>KKYV</td><td></td><td> 631</td>

<td>SILauN</td><td></td><td> 632</td>

<td>LauKKYL</td><td></td><td> 633</td>

<td>CapKKYL</td><td></td><td> 634</td>

<td>KYL</td><td></td><td>NO</td>

<td>KYL</td><td></td><td>NO</td>

<td>KKYNle</td><td></td><td> 635</td>

<td>VKKYL</td><td></td><td> 636</td>

<td>LNSILN</td><td></td><td> 637</td>

<td>YLNSILN</td><td></td><td> 638</td>

<td>KKYLNle</td><td></td><td> 639</td>

<td>KKYLN</td><td></td><td> 640</td>

<td>KKYLNS</td><td></td><td> 641</td>

<td>KKYLNSI</td><td></td><td> 642</td>

<td>KKYLNSIL</td><td></td><td> 643</td>

<td>KKKYLD</td><td></td><td> 644</td>

<td>Cyclic CKKYLC</td><td></td><td> 645</td>

<td>CKKYLK | | S-CH2-CO</td><td></td><td> 646</td>

PL 211 164 B1 cont. table 12

<td> 1</td><td> 2</td>

<td>KKYA</td><td> 647</td>

<td>WWTDTGLW</td><td> 648</td>

<td>WWTDDGLW</td><td> 649</td>

<td>WWDTRGLWVWTI</td><td> 650</td>

<td>FWGNDGIWLESG</td><td> 651</td>

<td>DWDQFGLWRGAA</td><td> 652</td>

<td>RWDDNGLWVVVL</td><td> 653</td>

<td>SGMWSHYGIWMG</td><td> 654</td>

<td>GGRWDQAGLWVA</td><td> 655</td>

<td>KLWSEQGIWMGE</td><td> 656</td>

<td>CWSMHGLWLC</td><td> 657</td>

<td>GCWDNTGIWVPC</td><td> 658</td>

<td>DWDTRGLWVY</td><td> 659</td>

<td>SLWDENGAWI</td><td> 660</td>

<td>KWDDRGLWMH</td><td> 661</td>

<td>QAWNERGLWT</td><td> 662</td>

<td>QWDTRGLWVA</td><td> 663</td>

<td>WNVHGIWQE</td><td> 664</td>

<td>SWDTRGLWVE</td><td> 665</td>

<td>DWDTRGLWVA</td><td> 666</td>

<td>SWGRDGLWIE</td><td> 667</td>

<td>EWTDNGLWAL</td><td> 668</td>

<td>SWDEKGLWSA</td><td> 669</td>

<td>SWDSSGLWMD</td><td> 670</td>

Table 13 - Sequences of the peptide Mdm / hdm antagonists

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>TFSDLW</td><td> 130</td>

<td>QETFSDLWKLLP</td><td> 131</td>

<td>QPTFSDLWKLLP</td><td> 132</td>

<td>QETFSDYWKLLP</td><td> 133</td>

<td>QPTFSDYWKLLP</td><td> 134</td>

PL 211 164 B1 cont. table 13

<td> 1</td><td> 2</td>

<td>MPRFMDYWEGLN</td><td> 135</td>

<td>VQNFIDYWTQQF</td><td> 136</td>

<td>TGPAFTHYWATF</td><td> 137</td>

<td>IDRAPTFRDHWFALV</td><td> 138</td>

<td>PRPALVFADYWETLY</td><td> 139</td>

<td>PAFSRFWSDLSAGAH</td><td> 140</td>

<td>PAFSRFWSKLSAGAH</td><td> 141</td>

<td>PXFXDYWXXL</td><td> 142</td>

<td>QETFSDLWKLLP</td><td> 143</td>

<td>QPTFSDLWKLLP</td><td> 144</td>

<td>QETFSDYWKLLP</td><td> 145</td>

<td>QPTFSDYWKLLP</td><td> 146</td>

Table 14 - Sequences of the Calmodulin antagonists peptide

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>SCVKWGKKEFCGS</td><td> 164</td>

<td>SCWKYWGKECGS</td><td> 165</td>

<td>SCYEWGKLRWCGS</td><td> 166</td>

<td>SCLRWGKWSNCGS</td><td> 167</td>

<td>SCWRWGKYQICGS</td><td> 168</td>

<td>SCVSWGALKLCGS</td><td> 169</td>

<td>SCIRWGQNTFCGS</td><td> 170</td>

<td>SCWQWGNLKICGS</td><td> 171</td>

<td>SCVRWGQLSICGS</td><td> 172</td>

<td>LKKFNARRKLKGAILTTMLAK</td><td> 173</td>

<td>RRWKKNFIAVSAANRFKK</td><td> 174</td>

<td>RKWQKTGHAVRAIGRLSS</td><td> 175</td>

<td>INLKALAALAKKIL</td><td> 176</td>

<td>KIWSILAPLGTTLVKLVA</td><td> 177</td>

<td>LKKLLKLLKKLLKL</td><td> 178</td>

<td>LKWKKLLKLLKKLLKKLL</td><td> 179</td>

PL 211 164 B1 cont. table 14

<td> 1</td><td> 2</td>

<td>AEWPSLTEIKTLSHFSV</td><td> 180</td>

<td>AEWPSPTRVISTTYFGS</td><td> 181</td>

<td>AELAHWPPVKTVLRSFT</td><td> 182</td>

<td>AEGSWLQLLNLMKQMNN</td><td> 183</td>

<td>AEWPSLTEIK</td><td> 184</td>

Table 15 - Sequences of Mast cell antagonist peptide / Mast cell protease inhibitors

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td>SGSGVLKRPLPILPVTR</td><td> 272</td>

<td>RWLSSRPLPPLPLPPRT</td><td> 273</td>

<td>GSGSYDTLALPSLPLHPMSS</td><td> 274</td>

<td>GSGSYDTRALPSLPLHPMSS</td><td> 275</td>

<td>GSGSSGVTMYPKLPPHWSMA</td><td> 276</td>

<td>GSGSSGVRMYPKLPPHWSMA</td><td> 277</td>

<td>GSGSSSMRMVPTIPGSAKHG</td><td> 278</td>

<td>RNR</td><td>NO</td>

<td>QT</td><td>NO</td>

<td>RQK</td><td>NO</td>

<td>NRQ</td><td>NO</td>

<td>RQK</td><td>NO</td>

<td>RNRQKT</td><td> 436</td>

<td>RNRQ</td><td> 437</td>

<td>RNRQK</td><td> 438</td>

<td>NRQKT</td><td> 439</td>

<td>RQKT</td><td> 440</td>

Table 16 - Sequences of the SH3 antagonist peptide

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>RPLPPLP</td><td> 282</td>

<td>RELPPLP</td><td> 283</td>

<td>SPLPPLP</td><td> 284</td>

<td>GPLPPLP</td><td> 285</td>

PL 211 164 B1 cont. table 16

<td> 1</td><td> 2</td>

<td>RPLPIPP</td><td> 286</td>

<td>RPLPIPP</td><td> 287</td>

<td>RRLPPTP</td><td> 288</td>

<td>RQLPPTP</td><td> 289</td>

<td>RPLPSRP</td><td> 290</td>

<td>RPLPTRP</td><td> 291</td>

<td>SRLPPLP</td><td> 292</td>

<td>RALPSPP</td><td> 293</td>

<td>RRLPRTP</td><td> 294</td>

<td>RPVPPIT</td><td> 295</td>

<td>ILAPPVP</td><td> 296</td>

<td>RPLPMLP</td><td> 297</td>

<td>RPLPILP</td><td> 298</td>

<td>RPLPSLP</td><td> 299</td>

<td>RPLPSLP</td><td> 300</td>

<td>RPLPMIP</td><td> 301</td>

<td>RPLPLIP</td><td> 302</td>

<td>RPLPPTP</td><td> 303</td>

<td>RSLPPLP</td><td> 304</td>

<td>RPQPPPP</td><td> 305</td>

<td>RQLPIPP</td><td> 306</td>

<td>XXXRPLPPLPXP</td><td> 307</td>

<td>XXXRPLPPIPXX</td><td> 308</td>

<td>XXXRPLPPLPXX</td><td> 309</td>

<td>RXXRPLPPLPXP</td><td> 310</td>

<td>RXXRPLPPLPPP</td><td> 311</td>

<td>PPPYPPPPIPXX</td><td> 312</td>

<td>PPPYPPPPYPXX</td><td> 313</td>

<td>LXXRPLPXTP</td><td> 314</td>

<td>'PXXRPLPXLP</td><td> 315</td>

<td>PPXΘXPPPΨP</td><td> 316</td>

PL 211 164 B1 cont. table 16

<td> 1</td><td> 2</td>

<td>+ PP'PPXKPXWL</td><td> 317</td>

<td>RPX&gt; PP_lpR + SXP</td><td> 318</td>

<td>PPVPPRPXXTL</td><td> 319</td>

<td>.pPtpLPipK</td><td> 320</td>

<td>+ ΘDXPLPXLP</td><td> 321</td>

Table 17 - Sequences of somatostatin or cortistatin mimetic peptides

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td>X¹-X²-Asn-Phe-Phe-Trp-Lys-Thr-Phe-X³-Ser-X⁴</td><td> 473</td>

<td>Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys</td><td> 474</td>

<td>Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys</td><td> 475</td>

<td>Cys Arg Asn Phe Phe T rp Lys Thr Phe Ser Ser Cys Lys</td><td> 476</td>

<td>Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys</td><td> 477</td>

<td>Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys</td><td> 478</td>

<td>Cys Arg Asn Phe Phe T rp Lys Thr Phe Ser Ser Cys</td><td> 479</td>

<td>Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys</td><td> 480</td>

<td>Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys</td><td> 481</td>

<td>Cys Lys Asn Phe Phe T rp Lys Thr Phe Ser Ser Cys Lys</td><td> 482</td>

<td>Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys</td><td> 483</td>

<td>Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys</td><td> 484</td>

<td>Cys Lys Asn Phe Phe T rp Lys Thr Phe Ser Ser Cys</td><td> 485</td>

<td>Asp Arg Met Pro Cys Arg Asn Phe Phe T rp Lys Thr Phe Thr Ser Cys Lys</td><td> 486</td>

<td>Met Pro Cys Arg Asn Phe Phe T rp Lys Thr Phe Thr Ser Cys Lys</td><td> 487</td>

<td>Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys</td><td> 488</td>

<td>Asp Arg Met Pro Cys Arg Asn Phe Phe T rp Lys Thr Phe Thr Ser Cys</td><td> 489</td>

<td>Met Pro Cys Arg Asn Phe Phe T rp Lys Thr Phe Thr Ser Cys</td><td> 490</td>

<td>Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys</td><td> 491</td>

<td>Asp Arg Met Pro Cys Lys Asn Phe Phe T rp Lys Thr Phe Thr Ser Cys Lys</td><td> 492</td>

<td>Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys</td><td> 493</td>

<td>Cys Lys Asn Phe Phe T rp Lys Thr Phe Thr Ser Cys Lys</td><td> 494</td>

<td>Asp Arg Met Pro Cys Lys Asn Phe Phe T rp Lys Thr Phe Thr Ser Cys</td><td> 495</td>

<td>Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys</td><td> 496</td>

<td>Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys</td><td> 497</td>

PL 211 164 B1

Table 18 - Sequences of peptide UKR antagonists

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td>AEPMPHSLNFSQYLWYT</td><td> 196</td>

<td>AEHTYSSLWDTYSPLAF</td><td> 197</td>

<td>AELDLWMRHYPLSFSNR</td><td> 198</td>

<td>AESSLWTRYAWPSMPSY</td><td> 199</td>

<td>AEWHPGLSFGSYLWSKT</td><td> 200</td>

<td>AEPALLNWSFFFNPGLH</td><td> 201</td>

<td>AEWSFYNLHLPEPQTIF</td><td> 202</td>

<td>AEPLDLWSLYSLPPLAM</td><td> 203</td>

<td>AEPTLWQLYQFPLRLSG</td><td> 204</td>

<td>AEISFSELMWLRSTPAF</td><td> 205</td>

<td>AELSEADLWTTWFGMGS</td><td> 206</td>

<td>AESSLWRIFSPSALMMS</td><td> 207</td>

<td>AESLPTLTSILWGKESV</td><td> 208</td>

<td>AETLFMDLWHDKHILLT</td><td> 209</td>

<td>AEILNFPLWHEPLWSTE</td><td> 210</td>

<td>AESQTGTLNTLFWNTLR</td><td> 211</td>

<td>AEPVYQYELDSYLRSYY</td><td> 430</td>

<td>AELDLSTFYDIQYLLRT</td><td> 431</td>

<td>AEFFKLGPNGYVYLHSA</td><td> 432</td>

<td>FKLXXXGYVYL</td><td> 433</td>

<td>AESTYHHLSLGYMYTLN</td><td> 434</td>

<td>YHXLXXGYMYT</td><td> 435</td>

Table 19 - Sequences of macrophage and / or T cell inhibitory peptides

<td>Sequence / Structure</td><td>SEQ ID NO:</td>

<td> 1</td><td> 2</td>

<td>Xaa-Yaa-Arg</td><td>NO</td>

<td>Arg-Yaa-Xaa</td><td>NO</td>

<td>Xaa-Arg-Yaa</td><td>NO</td>

<td>Yaa-Arg-Xaa</td><td>NO</td>

<td>Ala-Arg</td><td>NO</td>

<td>Arg-Arg</td><td>NO</td>

PL 211 164 B1 cont. table 19

<td> 1</td><td> 2</td>

<td>Asn-Arg</td><td>NO</td>

<td>Asp-Arg</td><td>NO</td>

<td>Cys-Arg</td><td>NO</td>

<td>Gln-Arg</td><td>NO</td>

<td>Glu-Arg</td><td>NO</td>

<td>Gly-Arg</td><td>NO</td>

<td>His-arg</td><td>NO</td>

<td>Ile-Arg</td><td>NO</td>

<td>Leu-Arg</td><td>NO</td>

<td>Lys-Arg</td><td>NO</td>

<td>Met-Arg</td><td>NO</td>

<td>Phe-Arg</td><td>NO</td>

<td>Ser-Arg</td><td>NO</td>

<td>Thr-Arg</td><td>NO</td>

<td>T rp-Arg</td><td>NO</td>

<td>Tire-Arg</td><td>NO</td>

<td>Val-Arg</td><td>NO</td>

<td>Ala-Glu-Arg</td><td>NO</td>

<td>Arg-Glu-Arg</td><td>NO</td>

<td>Asn-Glu-Arg</td><td>NO</td>

<td>Asp-Glu-Arg</td><td>NO</td>

<td>Cys-Glu-Arg</td><td>NO</td>

<td>Gln-Glu-Arg</td><td>NO</td>

<td>Glu-Glu-Arg</td><td>NO</td>

<td>Gly-Glu-Arg</td><td>NO</td>

<td>His-Glu-Arg</td><td>NO</td>

<td>Ile-Glu-Arg</td><td>NO</td>

<td>Leu-Glu-Arg</td><td>NO</td>

<td>Lys-Glu-Arg</td><td>NO</td>

<td>Met-Glu-Arg</td><td>NO</td>

<td>Phe-Glu-Arg</td><td>NO</td>

PL 211 164 B1 cont. table 19

<td> 1</td><td> 2</td>

<td>Pro-Glu-Arg</td><td>NO</td>

<td>Ser-Glu-Arg</td><td>NO</td>

<td>Thr-Glu-Arg</td><td>NO</td>

<td>Trp-Glu-Arg</td><td>NO</td>

<td>Tyr-Glu-Arg</td><td>NO</td>

<td>Val-Glu-Arg</td><td>NO</td>

<td>Arg-Ala</td><td>NO</td>

<td>Arg-Asp</td><td>NO</td>

<td>Arg-Cys</td><td>NO</td>

<td>Arg-Gln</td><td>NO</td>

<td>Arg-Glu</td><td>NO</td>

<td>Arg-Gly</td><td>NO</td>

<td>Arg-His</td><td>NO</td>

<td>Arg-Ile</td><td>NO</td>

<td>Arg-Leu</td><td>NO</td>

<td>Arg-Lys</td><td>NO</td>

<td>Arg-Met</td><td>NO</td>

<td>Arg-Phe</td><td>NO</td>

<td>Arg-Pro</td><td>NO</td>

<td>Arg-Ser</td><td>NO</td>

<td>Arg-Thr</td><td>NO</td>

<td>Arg-T rp</td><td>NO</td>

<td>Arg-Tyr</td><td>NO</td>

<td>Arg-Val</td><td>NO</td>

<td>Arg-Glu-Ala</td><td>NO</td>

<td>Arg-Glu-Asn</td><td>NO</td>

<td>Arg-Glu-Asp</td><td>NO</td>

<td>Arg-Glu-Cys</td><td>NO</td>

<td>Arg-Glu-Gln</td><td>NO</td>

<td>Arg-Glu-Glu</td><td>NO</td>

<td>Arg-Glu-Gly</td><td>NO</td>

PL 211 164 B1 cont. table 19

<td> 1</td><td> 2</td>

<td>Arg-Glu-His</td><td>NO</td>

<td>Arg-Glu-lle</td><td>NO</td>

<td>Arg-Glu-Leu</td><td>NO</td>

<td>Arg-Glu-Lys</td><td>NO</td>

<td>Arg-Glu-Met</td><td>NO</td>

<td>Arg-Glu-Phe</td><td>NO</td>

<td>Arg-Glu-Pro</td><td>NO</td>

<td>Arg-Glu-Ser</td><td>NO</td>

<td>Arg-Glu-Thr</td><td>NO</td>

<td>Arg-Glu-Trp</td><td>NO</td>

<td>Arg-Glu-Tyr</td><td>NO</td>

<td>Arg-Glu-Val</td><td>NO</td>

<td>Ala-Arg-Glu</td><td>NO</td>

<td>Arg-Arg-Glu</td><td>NO</td>

<td>Asn-Arg-Glu</td><td>NO</td>

<td>Asp-Arg-Glu</td><td>NO</td>

<td>Cys-Arg-Glu</td><td>NO</td>

<td>Gln-Arg-Glu</td><td>NO</td>

<td>Glu-Arg-Glu</td><td>NO</td>

<td>Gly-Arg-Glu</td><td>NO</td>

<td>His-Arg-Glu</td><td>NO</td>

<td>Ile-Arg-Glu</td><td>NO</td>

<td>Leu-Arg-Glu</td><td>NO</td>

<td>Lys-Arg-Glu</td><td>NO</td>

<td>Met-Arg-Glu</td><td>NO</td>

<td>Phe-Arg-Glu</td><td>NO</td>

<td>Pro-Arg-Glu</td><td>NO</td>

<td>Ser-Arg-Glu</td><td>NO</td>

<td>Thr-Arg-Glu</td><td>NO</td>

<td>Trp-Arg-Glu</td><td>NO</td>

<td>Tyr-Arg-Glu</td><td>NO</td>

PL 211 164 B1 cont. table 19

<td> 1</td><td> 2</td>

<td>Val-Arg-Glu</td><td>NO</td>

<td>Glu-Arg-Ala,</td><td>NO</td>

<td>Glu-Arg-Arg</td><td>NO</td>

<td>Glu-Arg-Asn</td><td>NO</td>

<td>Glu-Arg-Asp</td><td>NO</td>

<td>Glu-Arg-Cys</td><td>NO</td>

<td>Glu-Arg-Gln</td><td>NO</td>

<td>Glu-Arg-Gly</td><td>NO</td>

<td>Glu-Arg-His</td><td>NO</td>

<td>Glu-Arg-Ile</td><td>NO</td>

<td>Glu-Arg-Leu</td><td>NO</td>

<td>Glu-Arg-Lys</td><td>NO</td>

<td>Glu-Arg-Met</td><td>NO</td>

<td>Glu-Arg-Phe</td><td>NO</td>

<td>Glu-Arg-Pro</td><td>NO</td>

<td>Glu-Arg-Ser</td><td>NO</td>

<td>Glu-Arg-Thr</td><td>NO</td>

<td>Glu-Arg-Trp</td><td>NO</td>

<td>Glu-Arg-Tyr</td><td>NO</td>

<td>Glu-Arg-Val</td><td>NO</td>

Table 20 - Further exemplary pharmacologically active peptides

<td>Sequence / Structure</td><td>SEQ ID NO:</td><td>Activity</td>

<td> 1</td><td> 2</td><td> 3</td>

<td>VEPNCDIHVMWEWECFERL</td><td> 1027</td><td>a VEGF antagonist</td>

<td>GERWCFDGPLTWVCGEES</td><td> 1084</td><td>a VEGF antagonist</td>

<td>RGWVEICVADDNGMCVTEAQ</td><td> 1085</td><td>a VEGF antagonist</td>

<td>GWDECDVARMWEWECFAGV</td><td> 1086</td><td>a VEGF antagonist</td>

<td>GERWCFDGPRAWVCGWEI</td><td> 501</td><td>a VEGF antagonist</td>

<td>EELWCFDGPRAWVCGYVK</td><td> 502</td><td>a VEGF antagonist</td>

<td>RGWVEICAADDYGRCLTEAQ</td><td> 1031</td><td>a VEGF antagonist</td>

<td>RGWVEICESDVWGRCL</td><td> 1087</td><td>a VEGF antagonist</td>

<td>RGWVEICESDVWGRCL</td><td> 1088</td><td>a VEGF antagonist</td>

<td>GGNECDIARMWEWECFERL</td><td> 1089</td><td>a VEGF antagonist</td>

PL 211 164 B1 cont. table 20

<td> 1</td><td> 2</td><td> 3</td>

<td>RGWVEICAADDYGRCL</td><td> 1090</td><td>a VEGF antagonist</td>

<td>CTTHWGFTLC</td><td> 1028</td><td>an MMP inhibitor</td>

<td>CLRSGXGC</td><td> 1091</td><td>an MMP inhibitor</td>

<td>CXXHWGFXXC</td><td> 1092</td><td>MMP inhibitor</td>

<td>CXPXC</td><td> 1093</td><td>MMP inhibitor</td>

<td>CRRHWGFEFC</td><td> 1094</td><td>MMP inhibitor</td>

<td>STTHWGFTLS</td><td> 1095</td><td>MMP inhibitor</td>

<td>CSLHWGFWWC</td><td> 1096</td><td>CTLA4-mimetic</td>

<td>GFVCSGIFAVGVGRC</td><td> 125</td><td>CTLA4-mimetic</td>

<td>APGVRLGCAVLGRYC</td><td> 126</td><td>CTLA4-mimetic</td>

<td>LLGRMK</td><td> 105</td><td>Antiviral (HBV)</td>

<td>ICVVQDWGHHRCTAGHMANLTSHASAI</td><td> 127</td><td>C3b antagonist</td>

<td>ICVVQDWGHHRCT</td><td> 128</td><td>C3b antagonist</td>

<td>CVVQDWGHHAC</td><td> 129</td><td>C3b antagonist</td>

<td>STGGFDDVYDWARGVSSALTTTLVATR</td><td> 185</td><td>Vinculin binding</td>

<td>STGGFDDVYDWARRVSSALTTTLVATR</td><td> 186</td><td>Vinculin binding</td>

<td>SRGVNFSEWLYDMSAAMKEASNVFPSRRSR</td><td> 187</td><td>Vinculin binding</td>

<td>SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR</td><td> 188</td><td>Vinculin binding</td>

<td>SSPSLYTQFLVNYESAATRIQDLLIASRPSR</td><td> 189</td><td>Vinculin binding</td>

<td>SSTGWVDLLGALQRAADATRTSIPPSLQNSR</td><td> 190</td><td>Vinculin binding</td>

<td>DVYTKKELIECARRVSEK</td><td> 191</td><td>Vinculin binding</td>

<td>EKGSYYPGSGIAQFHIDYNNVS</td><td> 192</td><td>Binding C4BP</td>

<td>SGIAQFHIDYNNVSSAEGWHVN</td><td> 193</td><td>Binding C4BP</td>

<td>LVTVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVN</td><td> 194</td><td>Binding C4BP</td>

<td>SGIAQFHIDYNNVS</td><td> 195</td><td>Binding C4BP</td>

<td>LLGRMK</td><td> 279</td><td>Anti-HBV</td>

<td>ALLGRMKG</td><td> 280</td><td>Anti-HBV</td>

<td>LDPAFR</td><td> 281</td><td>Anti-HBV</td>

<td>CXXRGDC</td><td> 322</td><td>Inhibition of platelet aggregation</td>

<td>RPLPPLP</td><td> 323</td><td>Src antagonist</td>

<td>PPVPPR</td><td> 324</td><td>Src antagonist</td>

<td>XFXDXWXXLXX</td><td> 325</td><td>Anti-cancer (especially against sarcoma)</td>

<td>KACRRLFGPVDSEQLSRDCD</td><td> 326</td><td>p16-mimetic</td>

<td>RERWNFDFVTETPLEGDFAW</td><td> 327</td><td>p16-mimetic</td>

<td>KRRQTSMTDFYHSKRRLIFS</td><td> 328</td><td>p16-mimetic</td>

<td>TSMTDFYHSKRRLIFSKRKP</td><td> 329</td><td>p16-mimetic</td>

PL 211 164 B1 cont. table 20

<td> 1</td><td> 2</td><td> 3</td>

<td>RRLIF</td><td> 330</td><td>p16-mimetic</td>

<td>KRRQTSATDFYHSKRRLIFSRQIKIWFQNYYMKWKK</td><td> 331</td><td>p16-mimetic</td>

<td>KRRLIFSKRQIKIWFQNRYMKWKK</td><td> 332</td><td>p16-mimetic</td>

<td>Asn Gln Gly Arg His Phe Cys Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr Ala Ala Ser Cys Phe Gln</td><td> 498</td><td>CAP37-mimetic / binding LPS</td>

<td>Arg His Phe Cys Gly Gly Ala Leu Ile His Ala Arg Phe Val Met Thr Ala Ala Ser Cys</td><td> 499</td><td>CAP3-mimetic / binding LPS</td>

<td>Gly Thr Arg Cys Gln Val Ala Gly Trp Gly Ser Gln Arg Ser Gly Gly Arg Leu Ser Arg Phe Pro Arg Phe Val Asn Val</td><td> 500</td><td>CAP37 mimetic / LPS binding</td>

<td>WHWRHRIPLQLAAGR</td><td> 1097</td><td>Carbohydrate (GD1 alpha) -mimetic</td>

<td>LKTPRV</td><td> 1098</td><td>Binding e2GPI Ab</td>

<td>NTLKTPRV</td><td> 1099</td><td>Binding e2GPI Ab</td>

<td>NTLKTPRVGGC</td><td> 1100</td><td>Binding e2GPI Ab</td>

<td>KDKATF</td><td> 1101</td><td>Binding e2GPI Ab</td>

<td>KDKATFGCHD</td><td> 1102</td><td>Binding e2GPI Ab</td>

<td>KDKATFGCHDGC</td><td> 1103</td><td>Binding e2GPI Ab</td>

<td>TLRVYK</td><td> 1104</td><td>Binding e2GPI Ab</td>

<td>ATLRVYKGG</td><td> 1105</td><td>Binding e2GPI Ab</td>

<td>CATLRVYKGG</td><td> 1106</td><td>Binding e2GPI Ab</td>

<td>INLKALAALAKKIL</td><td> 1107</td><td>Membranowo Transporting</td>

<td>GWT</td><td>NO</td><td>Membranowo Transporting</td>

<td>GWTLNSAGYLLG</td><td> 1108</td><td>Membranowo Transporting</td>

<td>GWTLNSAGYLLGKINLKALAALAKKIL</td><td> 1109</td><td>Membranowo Transporting</td>

The present invention is also particularly useful for peptides having activity in the treatment of:

Cancer, where the peptide is a VEGF mimetic or a VEGF receptor antagonist, HER2 agonist or antagonist, CD20 antagonist and the like;

• asthma when the protein concerned is a CKR3 antagonist, an IL-5 receptor antagonist and the like;

• thrombosis when the protein concerned is a GPIIb antagonist, a GPIIIa antagonist and the like;

• autoimmune diseases and other conditions involving immune modulation, where 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.

Substrates. The present invention requires the presence of at least one substrate (F, F) attached to the peptide via the N-terminus, C-terminus or via the outer chain of one of the amino acid residues. Multiple substrates can also be used; for example, Fc residues at each end or Fc residues at one end and PEG residues at the other end or outer chain.

The Fc domain is a preferred substrate. The Fc domain may be fused to the N- or C-terminus of the peptide, or to both the N- and C-terminus. For TPO-mimetic peptides, domain containing molecules

PL 211 164 B1

Fc fused to the N-terminus of the peptide portion of the molecule are more bioactive than other such fusions, so fusions to the N-terminus are preferred.

As noted above, Fc variants are suitable media within the scope of the present invention. The native Fc can be extensively modified to form an Fc variant in accordance with the present invention, as long as binding to the "salvage" receptor is preserved; see, for example, WO 97/34631 and WO 96/32478. In such Fc variants, one or more native Fc sites can be deleted that provide structural features or functional activity not required for the fusion molecules of the invention. You can remove these sites by, for example, substituting or removing residues, inserting residues into place, or truncating portions containing a site. The inserted or substituted residues can also be altered amino acids, such as peptidomimetic or D-amino acids. Fc variants can be desirable for a number of reasons, several of which have been described above. Exemplary Fc variants include molecules and sequences in which:

1. Sites involved in disulfide bond formation have been removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cells used to produce the molecule of the invention. For this purpose, a cysteine-containing segment at the N-terminus may be truncated or the cysteine ​​residues may be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, a 20-amino acid segment from the N-terminus of SEQ ID NO: 2 may be truncated, or the cysteine ​​residues at positions 7 and 10 in SEQ ID NO: 2 may be deleted or substituted. Even when cysteine ​​residues are removed, one chain of the Fc domain may in they continue to create a dimeric Fc domain that sticks together non-covalently.

2. The native Fc is modified making it more compatible with the chosen host cell. For example, one can remove the PA sequence close to the N-terminus of a typical native Fc that can be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. An N-terminal methionine residue may also be added, especially when the molecule is recombinantly expressed in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 2 (Fig. 4) is one such Fc variant.

3. The N-terminal portion of the native Fc is removed, preventing N-terminal heterogeneity, when expressed in a selected host cell. To this end, any of the first 20 amino acid residues at the N-terminus may be removed, especially those at positions 1, 2, 3, 4, and 5.

4. One or more glycosylation sites are removed. Residues that are of the glycosylated type (e.g., asparagine) may confer the capacity for a cytolytic response. Such residues can be removed or substituted with non-glycosylated residues (for example, alanine).

5. Sites involved in interaction with the compliment, such as the C1q binding site, have been removed. For example, the EKK sequence of human IgG1 can be deleted or substituted. Recruitment of complement may not be beneficial to the molecules of the invention and such Fc variant should therefore be avoided.

6. Sites that affect binding to Fc repceptors other than the "salvage" receptor have been removed. The 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 may also be removed.

7. ADCC site removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) regarding ADCC sites in IgG1. These sites are also not required for the fusion molecules of the present invention and may also be removed.

8. When the native Fc is derived from a non-human antibody, the native Fc can be humanized. Typically, humanizing a native Fc, one will substitute selected residues in the non-human native Fc with residues that would normally be found in native human Fc. Techniques for humanizing antibodies are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 2 (Fig. 4) the leucine at position 15 may be substituted with glutamate; glutamate at position 99, alanine; and lysines at positions 101 and 103 with alanines. In addition, one or more tyrosine residues may be replaced by phenylalanine residues.

An alternative medium would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (for example, a peptidomimetic compound) capable of binding to a "salvage" receptor. For example, it can be used as a polypeptide substrate as described herein

U.S. Patent No. 5,739,277, issued April 14, 1998: Presta et al. Peptides could also be selected by displaying the phage for binding to the "salvage" FcRn receptor. Such "salvage" receptor binding compounds are also encompassed by the term "vehicle" and are within the scope of the present invention. Such media should be selected to increase half-life (for example, by protease avoidance sequences) and to reduce immunogenicity (for example, by non-immunogenicity promoting sequences as found in antibody humanization).

As noted above, polymeric substrates can also be used as F¹ and F². Various means for attaching chemical moieties used as substrates are currently available, see, for example. Patent Cooperation Treaty ("PCT") International Publication No. WO 96/11953 entitled "Compositions and Methods for Chemical N-Terminal Modification of Proteins" incorporated herein by reference. This PCT publication discloses, inter alia, the selective attachment of water-soluble polymers to the N-terminus of proteins.

A preferred polymeric support is polyethylene glycol (PEG). The PEG group may be one of any suitable molecular weight and may be linear or branched. The average molecular weight of the PEG (groups) 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. PEG groups will generally be attached to compounds of the invention by acylation or reductive alkylation via a reactive group from a PEG residue (e.g., aldehyde, amino, thiol, or ester group) to a reactive group in the compound under consideration (e.g., aldehyde, amino, or ester group) ).

A useful PEGylation strategy for synthetic peptides involves combining, by formation of a conjugate bond in solution, the peptide and PEG residues, each having a particular reaction function for the other. Peptides can readily be made by conventional solid phase syntheses (see, for example, Figs. 5 and 6 and accompanying text). Peptides are "pre-activated" with an appropriate group at a specific site. The precursors are purified and fully characterized before reacting with the PEG moiety. Ligation of a peptide to PEG usually takes place in the aqueous phase and can be easily monitored by analytical reverse phase HPLC. PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser mass spectrometry.

Polysaccharide polymers are another type of water-soluble polymers that can be used to modify proteins. Dextrans are polysaccharide polymers comprising the individual glucose subunits predominantly linked by α1-6 linkages. As such, dextran is available in many types with different molecular weight ranges and is usually readily available in a form having a molecular weight of from about 1 kD to about 70 kD. Dextran is a suitable water-soluble polymer for use in the present invention as a substrate, either alone or in combination with another substrate (for example, Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has already been described; see, for example, European Patent Publication No. 0 315 456, which is incorporated herein 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.

Linkers. Any "linker" group is an optional group. When present, its chemical structure is not critical as it primarily acts as an expanding element. The linker preferably consists of amino acids linked by peptide bonds. Thus, in a preferred embodiment, the linker is formed 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 can be glycosylated as will be appreciated by those skilled in the art. In a more preferred embodiment, 1 to 20 amino acids are selected from Gly, Ala, Pro, Asn, Gln, and Lys. Even more preferably, the linker is made up of a majority of non-hindered amino acids such as glycine and alanine. Hence, preferred linkers are polyglycines (especially (Gly) 4, (Gly) 5), poly (Gly-Ala) and polyalanines. Other specific examples of linkers are:

(Gly) 3Lys (Gly) 4 (Gly) 3AsnGlySer (Gly) 2 (Gly) 3Cys (Gly) 4 and GlyProAsnGly (SEQ ID NO (SEQ ID NO (SEQ ID NO (SEQ ID NO

333) ;

334) ;

335) ;

336) .

PL 211 164 B1

To explain the above designations, for example, (Gly) 3Lys (Gly) 4 means Gly-Gly-Gly-Lys-Gly-GlyGly-Gly. Combinations of Gly and Ala are also preferred. Linkers shown are examples only;

The linkers encompassed by the present invention can be much longer and include other residues as well.

Non-peptide linkers are also possible. For example, alkyl linkers such as -HN- (CH2) s-CO-, where s = 2-20 can be used. These alkyl linkers may be further substituted with any non-hindered group such as lower alkyl (for example, C1-C6), lower acyl, halogen (for example Cl, Br), CN group, NH2 group, phenyl group, and the like. An exemplary non-peptide linker is a PEG linker

VI ο

<img file="PL211164B1_D0001.tif" />

where "n" is selected such that the linker has a molecular weight of from 100 to 5000 kD, preferably from 100 to 500 kD. Peptide linkers can be altered to derivatize in the same way as described above.

Derivatives. The inventors also contemplate derivatization of the peptide and / or the support portion of the present compounds. Such derivatives can improve the solubility, absorption, biological half-life and the like of the present compounds. The moieties may alternatively eliminate or attenuate the undesirable side effects of the compounds, etc. Exemplary derivatives include compounds in which:

1. The relationship or some part of it is cyclical. For example, the peptide portion can be modified to contain two or more Cys residues (for example, in the linker) which could be cyclized by forming a disulfide bond. Information on publications concerning cyclized derivatives is given in Table 2.

2. The compound is cross-linked or rendered capable of cross-linking between molecules. For example, a portion of a peptide can be modified to contain one Cys residue and thus be able to form an intermolecular disulfide hang with a similar molecule. The compound may also be cross-linked at its C-terminus as in the molecule shown below.

VII

1 ^H. AND

F.¹- (X¹)_b-CO-N ^ _A_NH2

F.¹- (X¹)_b-CO-N ^^ NH

3.

4. One or more peptidyl bonds [-C (O) NR-] are replaced by a non-peptidyl linkage. Examples of non-peptidyl combinations are: -CH2-carbamate [-CH2-OC- (O) NR-], phosphonate, -CH2-sulfonamide [-CH2-S (O) 2NR-], urea [-NHC (O) NH-], -CH2-tertiary amine, and the alkylated peptide [-C (O) NR⁶-, where R.⁶ means lower alkyl].

5. The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to provide a substituted amine. Exemplary N-terminal derivative groups include: -NRR¹ (other than -NH2), -NRC (O) R¹, -NRC (O) OR¹, -NRS (O) 2R¹, -NHC (O) NHR¹, succinimide or benzyloxycarbonyl-NH- (CBZ-NH-), where each of R and R¹ are 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, methods described in the art can be used to add (NH-CH2-CH2-NH2) 2 to compounds of the present invention having at the C-terminus any one of SEQ ID NOS: 504 to 508. Likewise, the methods described in in the art to add -NH2 to compounds of the present invention having any of se56 at the C-terminus

The sequence of SEQ ID NOS: 924 to 955, 963 to 972, 1005 to 1013, or 1018 to 1023.

C-terminal derivative groups include, for example, -C (O) R² where r² is a lower group or

-NR³R⁴ where r³ and R.⁴ are independently hydrogen or C1-C8 alkyl (preferably C1-C4 alkyl).

7. The disulfide bond is replaced with another, preferably more stable, cross-linking moiety (for example, an alkylene). See, for example, Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.

8. One or more individual amino acid residues are modified. Various derivatizing agents are known to react specifically with selected side chains or terminal residues as described in detail below.

Lysinyl residues and amino terminal residues can be reacted with succinic anhydride or other carboxylic acid anhydrides which result in charge reversal of the lysinyl residues. Other suitable reagents for derivatizing residues with alpha-amino groups include imidoesters such as: methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzene sulfonic acid; O-methylisourea; 2,4 pentanedione and transaminase catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin, among others. Derivatization of arginine residues requires carrying out the reaction in an alkaline environment due to the high pKa of the guanidinyl functional group. Moreover, these reagents can react with the lysine groups as well as with the epsilon-amino residues of the arginine group.

Specific modifications of tyrosyl residues have been extensively studied, especially in terms of introducing spectral markers into tyrosyl residues by reaction with aromatic diazonium compounds or with tetranitromethane. N-acetylimidizole and tetranitromethane are most commonly used to prepare O-acetyl-tyrosyl compounds and 3-nitro derivatives, respectively.

The side chain carboxyl groups (aspartyl or glutamyl) can be selectively modified by reaction with carbodiimides (R'-N = C = N-R ') such as 1-cyclohexyl-3- (2-morpholinyl- (4-ethyl) -carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide In addition, aspartyl and glutamyl residues can be converted into aspartyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are often deaminated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues may be deaminated in a mildly acidic environment. Both forms of these groups are included in the present invention.

Cysteinyl residues may be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, for example, Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.

Derivatization with bifunctional reagents is useful in order to link - by cross-linking - peptides or functional derivatives thereof to a water-insoluble support matrix or other macromolecular supports. Commonly used cross-linking reagents are, for example: 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, 4-azidosalicylic acid esters, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3,3 '-dithiobis- (succinimidylpropionate) and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3 - [(p-azidophenyl) dithio] propioimidate lead to the formation of photoactivatable intermediates capable of forming crosslinks in the presence of light. Alternatively, reactive, water-insoluble matrices, such as cyanogen-activated carbohydrates and reactive substrates described in US Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537 and 4,330,440 can be used to immobilize proteins.

Carbohydrate (oligosaccharide) groups may conveniently be attached to sites which are known to be glycosylation sites in proteins. Generally, oxygen-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while oligosaccharides are attached via nitrogen to asparagine (Asn) residues when they are part of the Asn-X-Ser / Thr sequence, where X can be any amino acid except proline. Preferably "X" is one of the 19 naturally occurring amino acids, not including proline. The structures of the N-linked and O-visited oligosaccharides and sugar residues found in each type are different.

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One type of sugar that is commonly found in both is N-acetylneuraminic acid (called sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, through its negative charge, can impart acidic properties to the glycosylated compound. Such site (s) may be incorporated into a linker in the compounds of the present invention and are preferably glycosylated in the cell during recombinant production of the polypeptide compounds (for example, in mammalian cells such as CHO, BHK, COS). However, such sites may be further glycosylated by synthetic or semi-synthetic procedures known in the art.

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 alpha-amino groups on lysine, arginine and histidine side chains (Creighton, TE, Proteins: Structure and Molekule Properties, WH Freeman &amp; Co., San Francisco, pp. 79-8 (1983)).

The compounds of the present invention may also be altered on the DNA level. The DNA sequence of any portion of the compound may be changed to codons more compatible with the host cells of choice. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons may be substituted to eliminate restriction sites or include "silent" restriction sites that may be useful in processing DNA in the host cell of choice. The substrate, linker, and peptide DNA sequences may be modified to include any of the above-mentioned sequence changes.

Methods of execution

The compounds of the present invention can to a large extent also be produced in transformed host cells using recombinant DNA techniques. To do this, a recombinant DNA molecule encoding a peptide was generated. Methods for producing such DNA molecules are well known in the art. For example, the peptide coding sequences could be cleaved from the DNA using appropriate restriction enzymes. Alternatively, DNA molecules could be produced using chemical synthesis techniques such as the phosphoramidite method. Also, a combination of these techniques could be used.

The invention also includes a vector encoding peptides in a suitable host. The vector comprises a DNA molecule encoding peptides operably linked to appropriate expression control sequences. Methods for making this operative linkage either before or after introduction of a DNA molecule encoding a peptide into a vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, closing signals, polyadenylation signals, and other signals involved in the control of transcription and translation.

The resulting vector, including the DNA molecule encoding the peptide, is used to transform a suitable host. This transformation can be performed using methods well known in the art.

Any of the large number of available and well-known host cells can be used in the practice of the present invention. The choice of a particular host depends on a number of factors recognized in the art. These factors include, for example, compatibility with the selected expression vector, toxicity of the peptides encoded by the DNA molecule to host cells, rate of transformation, ease of isolation of the peptides, expression characteristics, biosafety, and cost. The balance of these factors must clash with the understanding that not all host cells may be equally effective at expressing a particular DNA sequence. As part of these general guidelines, useful microbial hosts are bacteria (such as E. coli sp.), Yeast (such as Saccharomyce sp.), And other fungi, insects, plants, mammalian (including human) cells, or other hosts. known in the art.

The transformed host is then cultured and purified. Host cells can be grown 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.

The present compounds can also be produced by synthetic methods. For example, solid phase synthesis techniques can be used. Suitable techniques are well known in the art and may include those described in Merrifield, Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem. Soc. 85: 2149 (1963); Davis et al., Biochem. Intl. 10: 39458

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414 (1985); Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Patent No.

3,941,763; Finn et al. (1976), The Proteins, (3rd ed.), Vol. 2, pp. 105-253 (1976); and Erickson et al.,

The Proteins, (3rd ed.), Vol. 2, pp. 257-527 (1976). Solid phase synthesis is the preferred technique for the production of individual peptides because it is the most cost-effective way to produce small peptides.

Compounds that contain derivatized peptides or that contain non-peptide groups can be synthesized by well known techniques of organic chemistry.

Applications of compounds

General. The compounds of the present invention exhibit pharmacological activity resulting from their ability to bind to a protein of interest as agonists, mimetics or antagonists of the native ligands of such considered proteins. The utility of particular compounds is given in Table 2. The activity of these compounds can be measured by assays known in the art. For TPO-mimetic and EPO-mimetic compounds, in vivo assays are further described in the Examples section below.

In addition to their therapeutic uses, the compounds of the present invention are useful in the diagnosis of diseases characterized by a dysfunction of the associated protein concerned. In a first embodiment, a method for detecting, in a biological sample, a protein of interest (e.g., a receptor) which is capable of being activated, comprising the steps of: (a) contacting the sample with a compound of the present invention; and (b) detecting activation of the protein of interest by the compound. Biological samples include tissue samples, intact cells, or extracts thereof. The compounds of the present invention can be used as part of a diagnostic kit for detecting the presence of the associated proteins of interest in a biological sample. Such kits use the compounds of the invention attached with a tag to enable detection. The compounds are useful for identifying the normal or abnormal proteins under consideration. In the case of EPOmimetic compounds, for example, the presence of an abnormal protein of interest in a biological sample may indicate disorders such as Diamond-Blackfan anemia where the EPO receptor is believed to be dysfunctional.

Therapeutic applications of EPO-mimetic compounds. The EPO-mimetic compounds of the invention are useful in the treatment of disorders characterized by low levels of red blood cells. The invention encompasses methods of modulating endogenous EPO receptor activity in a mammal, preferably methods of increasing EPO receptor activity. In general, any condition treatable with erythropoietin, such as anemia, can also be treated with the EPO-mimetic compounds of the invention. The compounds are administered in an amount and route of administration that is appropriate to the nature and severity of the condition being treated and can be determined by those skilled in the art. Administration by injection, subcutaneously, intramuscularly or intravenously, is preferred.

Therapeutic applications of TPO-mimetic compounds. For TPO-mimetic compounds, standard assays can be used, such as those described in WO 95/25746 entitled "Compositions and Methods for Stimulating Megakaryocyte Growth and Differentiation". In vivo trials are described below, including the examples.

Conditions to be treated by the methods and compositions of the invention are generally those that include the presence of a megakaryocyte / platelet deficiency or an expected or anticipated future megakaryocyte / platelet deficiency (for example, due to elective surgery or platelet delivery). Such conditions may be the consequence of a deficiency (temporary or continuous) of active Mpl ligand in vivo. The generic term for platelet deficiency is thrombocytopenia, and therefore the methods and compositions of the present invention are generally available for the prophylaxis or therapeutic treatment of thrombocytopenia in patients in need thereof.

Thrombocytopenia (platelet deficiency) can occur for a number of reasons, including chemotherapy and other medication treatments, radiation therapy, surgery, blood loss from accidents, and other specific medical conditions. Exemplary specific disease states that include thrombocytopenia and can be treated in accordance with the present invention are: aplastic anemia; idiopathic thrombocytopenia, metastatic tumors causing thrombocytopenia; systemic lupus erythematosus; splenomegaly; Fanconi syndrome; vitamin B12 deficiency; folic acid deficiency; May-Hegglin anomaly; Wiskott-Aldrich syndrome; paroxysmal nocturnal hemoglobinuria. Also, certain AIDS treatments cause thrombocytopenia (e.g., AZT). An increase in the number of platelets may be beneficial in certain disorders of wound healing.

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Due to the anticipation of a platelet deficiency, for example as a result of a future surgery, a compound of the present invention may be administered several days to several hours before the need for platelets. In acute cases, for example, accidental and / or significant blood loss, a compound of the present invention may be administered simultaneously with the blood or cleaned platelets.

The TPO-mimetic compounds of the present invention may also be used to stimulate certain types of cells other than megakaryocytes, if these cells are found to express the Mpl receptor. Conditions associated with such cells that express the Mpl receptor that respond to stimulation with the Mpl ligand are also encompassed by the present invention.

The TPO-mimetic compounds of the present invention can be used in any situation where the production of platelets or platelet precursor cells is desirable or where stimulation of the c-Mpl receptor is desirable. Accordingly, for example, the compounds of the present invention can be used to treat a variety of conditions in mammals where plaques, megakaryocytes, and the like are essential. Such conditions are described in detail in the following exemplary references: WO 95/26746; WO 95/21919; WO 95/18858; WO 95/21920, which are hereby referenced and incorporated herein.

The TPO-mimetic compounds of the present invention may also be used to maintain the viability or shelf life of platelets and / or megakaryocytes and related cells. Accordingly, an effective amount of one or more such compounds may be added to a composition containing such cells.

The therapeutic methods, compositions and compounds of the present invention can also be used, alone or in combination with other cytokines, soluble Mpl receptor, hematopoietic factors, interleukins, growth factors, or antibodies to treat disease states characterized by other symptoms as well as platelet deficiencies. It can be expected that the inventive compound will prove useful in the treatment of certain forms of thrombocytopenia in combination with general stimulants of haemopoiesis such as IL-3 or GM-CSF. Other megakaryocytic stimulating factors, i.e., meg-CSF, hematopoietic stem cell factor (SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or other molecules with megakaryocyte stimulating activity, can also be used with the Mpl ligand. Additional exemplary cytokines or hematopoietic factors for such co-administration include: lL-1 alpha, lL-1beta, lL-2, lL-3, ll-4, ll-5, ll-6, ll-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.

Furthermore, it is useful to administer either simultaneously or sequentially an effective amount of the soluble breast Mpl receptor, which appears to have the property of causing the megakaryocytes to fragment into platelets as soon as the megakaryocytes reach mature form. Thus, it is expected that administration of a compound of the present invention (to enhance the amount of mature megakaryocytes) followed by administration of the soluble Mpl receptor (to inactivate the ligand and allow the mature megakaryocyte to produce platelets) will prove to be a particularly effective means for stimulating platelet production. The dosages given above should be adjusted to compensate for these additional components in the therapeutic composition. The progress of the treated patient can be monitored by known methods.

In cases where compounds of the invention are added to platelet and / or megakaryocyte and related cell compositions, the amount to be incorporated into the composition will generally be determined experimentally using known techniques and trials. An exemplary range of these amounts is 0.1 μg - 1 mg of a compound of the invention per 10⁶ cells.

Pharmaceutical compositions

General. The present invention also includes methods of using the pharmaceutical compositions of the compounds of the invention. Such pharmaceutical compositions can be administered by injection or orally, nasally, transdermally, or by other forms of administration. Generally, pharmaceutical compositions comprising effective amounts of a compound of the invention are included in the present invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluents with different buffer content (e.g., Tris-HCl, acetates, phosphates), pH, and ionic strength; additives such as detergents and solubilizing agents (e.g. Tween 80, Polysorbate 80), antioxidants (e.g. ascorbic acid, sodium metabisulfite), preservatives (e.g. 60

For example, Thimersol, benzyl alcohol) and excipients (for example, lactose, mannitol); incorporation of the material into particles of preparations of polymeric compounds such as polylactonic acid, polyglycolic acid, etc. or for liposomes. Hyaluronic acid can also be used to extend the duration of circulation. Such compositions can affect the physical state, stability, rate of in vivo release, and degree of in vivo purity of the proteins and derivatives present. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, which are hereby incorporated by reference. The compositions may be in liquid form, or they may be in the form of a dry powder such as a lyophilisate. Sustained release implantable forms such as transdermal formulations are also envisaged.

Oral administration forms. Oral solid dosage forms are also contemplated herein for use as are generally described in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton PA 18042) in Chapter 89, which is hereby incorporated by reference. Solid forms include tablets, capsules, pills, troches or lozenges, sachets, or powders. Also, encapsulation in liposomes and protenoids may be used in preparing galenic forms of the present compositions (such as, for example, the proteinoid microcapsules described in US Patent No. 4,925,673). Encapsulation in liposomes can be used and liposomes can be derivatized with different polymers (for example, US Patent No. 5,013,556). For a description of possible solid forms for therapeutic purposes, see Marshall, K., Modern Pharmaceutics, Edited by GS Banker and CT Rhodes Chapter 10, 1979, incorporated herein by reference. In general, the composition will include the compounds of the invention and inert ingredients which will provide protection against stomach conditions and release of biologically active substances in the intestine.

Also, oral administration forms of the above compounds of the invention are specifically envisaged. If necessary, the compounds can be chemically modified to be effective when administered orally. Generally, a chemical modification is envisaged to attach at least one moiety to the present compound molecule, the moiety allowing (a) inhibition of proteolysis and (b) absorption into the bloodstream from the stomach or intestines. It is also desirable to increase the overall stability of the present compound and increase the circulation time in the body. Moieties useful as a covalently attached substrate in accordance with the present invention may also be used for this purpose. Examples of such residues include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, and polyproline (Abuchowski and Davis, Soluble Polimer-Enzyme Adducts, Enzymes as Drugs, Hocsenberg, &amp; Roberts, Interscience, New York, NY, (1981), pp. 367-383; Newmark, et al., J. Appl. Biochem. 4: 185-189 (1982)). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-thioxocane. Preferred residues for pharmaceutical use as indicated above are PEG residues.

In the case of the oral administration form, 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 the absorption of the therapeutic compounds of the present invention. The clinical efficacy of the heparin formulation using SNAC was demonstrated in a Phase II study by Emisphere Technologies. See US Patent No. 5,792,451 "Oral drug delivery composition and methods."

The compounds of the invention may be incorporated into the compositions as fine multiparticles in the form of granules or flakes with a grain size of about 1 mm. The form of the material for administration in capsules can also be a powder, lightly compressed lumps or even tablets. Medicines can be made by pressing.

Any colorants and fragrances can be included in the composition. For example, a composition may be prepared with the protein (or derivative) present, for example in the form of liposome capsules or microspheres, and then incorporated into a food product such as a cool drink containing colors and flavors.

You can dilute or increase the volume of the compound of the invention by using inert substances. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, cane sugar, modified dextrans, and starch. Certain inorganic salts can also be used as fillers, including calcium triphosphate, magnesium carbonate, and sodium chloride. Some commercially available thinners are: Fast-Flo, Emdex, STA-Rx 1500, Emcompress, and Avicell.

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Disintegrants can be included in the composition containing the therapeutic substance in a solid dosage unit form. Substances used as disintegrants include, but are not limited to, starch, including the market sten starch disintegrant, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, carboxymethylcellulose acid, natural sponge and bentonite can all be used. Another form of disintegrants are the insoluble cation exchange resins. Powdered gums can be used as disintegrants and binders and can include powdered gums such as agar, karaya, or tragacanth. Alginic acid and its sodium salt are also used as useful disintegrants.

Binders that can be used to keep the form of the drug compact and form a hard tablet include substances derived from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC), and carboxymethyl cellulose (CMC). Two compounds, polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) can be used in the form of alcoholic solutions to granulate the therapeutic agent.

Lubricants may be included in the therapeutic composition to prevent sticking during preparation of the composition. Lubricants can be used as a protective layer between the medicament and the wall of the nozzle and can include, but are not limited to: stearic acid and 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.

"Glidants" can also be added, which can improve the flow properties of the medicament during manufacture and aid displacement during compression. "Glidants" can include starch, talc, pyrogenic silica, and hydrated aluminosilicates.

A surfactant may be added as a wetting agent to increase the solubility of the medicament in the aqueous medium. Surfactants may include anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents may be used and could include benzalkonium chloride and benzethonium chloride. A list of potential nonionic detergents that could be added to the compositions as surfactants includes: lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene 10, 50 and 60 hydrogenated castor oil, glycerin monostearate, polysorbate 40, 60, 65 and 80, fatty acid ester and cane sugar, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the composition containing the protein or a derivative thereof either alone or in a mixture in varying proportions.

Additives that potentially increase the absorption of the present compound are, for example, fatty acids, oleic acid, linoleic acid, and linolenic acid.

Compositions with the controlled release of the drug substance may be desirable. The drug could be loaded into an internal matrix that allows it to be released by a diffusion or leaching mechanism of, for example, gum. Slowly degenerating matrices can also be a component of the composition, for example alginates, polysaccharides. Another form of controlled release of the drug substance present involves a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semi-permeable membrane that allows water to penetrate and push the drug through a single small opening due to osmotic effects. Some intestinal coatings also have a delayed release effect.

Other coatings can be used in the manufacture. These include various sugars that can be applied in the coating pan. The medicament can also be delivered as a film coated tablet, and the materials used in this case are divided into two groups. The first are non-enteric materials, including methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methyl hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxy methyl cellulose, providone and polyethylene glycol. The second is the gut materials, which are commonly esters of phthalic acid.

Mixtures of materials can be used to ensure optimal film coverage. Film coating can be carried out in a coating pan or in a fluidized bed, or by a compression coating method.

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Forms for pulmonary administration. Pulmonary administration of the proteins present (or their derivatives) is also envisaged. The protein (or derivative) is delivered to the lungs of the mammal by inhalation and passes through the pulmonary epithelial lining into the bloodstream. Other reports of this include: Adjei et al., Pharma. Res. 7: 565-569 (1990); Adjei et al., International Journal of Pharmaceutics 63: 135-144 (1990) (leuprolide acetate); Braquet et al., J. Cardiovasc. Pharmacol. 13 (suppl.5): p. 143-146 (1989) (Endothelin-1); Hubbard et al., Annals Int. Med. 3: 206-212 (1989) (a1-antitrypsin); Smith et al., J. Clin. Invest. 84: 1145-1146 (1989) (a1-proteinase); Oswein et al., "Aerosolization of Proteins" Proc. Symp. Resp. Drug Delivery II, Keystone, Colorado, March, 1990 (recombinant human growth hormone); Debs et al., J. Immunol. 140: 3482-3488 (1988) (interferon-γ and tumor necrosis factor TNF α) and Platz et al., US Patent No. 5,284,655 (granulocyte colony stimulating factor).

A wide variety of mechanical devices designed for the pulmonary delivery of therapeutic products are envisaged for practicing the present invention, including, but not limited to, nebulizers, dosing device inhalers, and dry powder inhalers, all known to those skilled in the art. Some specific examples of commercially available devices suitable for use in the practice of the present invention are: Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; a Ventolin dosing device inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina, and a Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.

All such devices require the use of compositions suitable for the release of the compounds of the invention. Typically, each composition is specific to the device used and may require the use of a suitable propellant in addition to diluents, adjuvants and / or carriers useful in therapy.

The compounds of the invention should most preferably be formulated in a fine particle form with an average grain size less than 10 µm (or microns), most preferably 0.5 to 5 µm, for most effective delivery to the peripheral regions of the lungs.

Pharmaceutically acceptable carriers may include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in the compositions may include DPPC, DOPE, DSPC, and DOPC. Natural or synthetic surfactants can be used. Polyethylene glycol can be used (even independently of its use in derivatization of the protein or its analog). Dextrans such as cyclodextran may also be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. The amino acids may be used as they are used in buffered compositions.

The use of liposomes, microcapsules or microspheres, inclusion complexes or other types of carriers is also envisaged.

Compositions suitable for use in a nebulizer, either by injection or ultrasonic, will typically contain the compounds of the invention dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The composition may also contain a buffer and a simple sugar (for example, for protein stabilization and regulation of osmotic pressure). The nebulizer composition may also contain a surfactant to reduce or prevent surface-induced protein aggregation caused by solution spraying during aerosol formation.

Compositions for use in an inhaler with a metering device will generally contain a finely divided powder containing a compound of the invention suspended in a nebulizer (propellant) with the aid of a surfactant. The propellant may be any conventional material used for this purpose such as chlorofluorocarbon, hydrochlorofluorocarbon, hydrofluorocarbon or hydrocarbon including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, or mixtures thereof. Suitable surfactants include sorbitan trioleate and soybean lecithin. Oleic acid may also be useful as a surfactant.

Compositions for release in a dry powder inhaler will include a finely divided dry powder containing a compound of the invention and may also include a bulking agent such as lactose, sorbitol, cane sugar, mannitol, trehalose, or xylitol in amounts that are

These compounds facilitate the release of the powder from the device, for example 50 to 90% by weight based on the weight of the composition.

Forms for donor administration. Intranasal administration of a compound of the invention is also envisaged. Intranasal administration allows the protein to enter the bloodstream immediately after administration of the therapeutic agent into the nose, without the product having to be deposited in the lungs. Compositions for intranasal administration include dextran or cyclodextran compositions. Administration by means of transmembrane transport across other mucous membranes is also contemplated.

Dosage. The dosage regimen for a method of treating the conditions indicated above will be determined by the attending physician taking into account a variety of drug modifying factors, for example, age, condition, weight, sex and diet of the patient, severity of infection, timing of administration, and other clinical factors. Generally, the dose should be in the range of 0.1 µg to 100 mg of the compound of the invention per kilogram of body weight per day, preferably 0.1 to 1000 µg / kg; more preferably 0.1 to 150 µg / kg,

Specific preferred embodiments

The inventors have established favorable peptide sequences for molecules having different kinds of activity. The researchers then identified the preferred structures of these preferred peptides linked to the preferred bonds and supports. The preferred structures for these preferred peptides are summarized in Table 21 below.

Table 21 - Preferred embodiments

<td>Sequence / Structure</td><td>SEQ ID NO:</td><td>Activity</td>

<td>F.¹- (G)₅-IEGPTLRQWLAARA- (G) 8-IEGPTLRQWLAARA</td><td> 337</td><td>TPO-mimetic</td>

<td>IEGPTLRQWLAARA- (G) 8-IEGPTLRQWLAARA- (G) sF¹</td><td> 338</td><td>TPO-mimetic</td>

<td>F.¹- (G) s-IEGPTLRQWLAARA</td><td> 1032</td><td>TPO-mimetic</td>

<td>IEGPTLRQWLAARA- (Gs) -F¹</td><td> 1033</td><td>TPO-mimetic</td>

<td>F.¹- (G) s-GGTYSCHFGPLTWVCKPQGG- (G) 4- GGTYSCHFGPLTWVCKPQGG</td><td> 339</td><td>EPO-mimetic</td>

<td>GGTYSCHFGPLTWVCKPQGG- (G) 4- GGTYSCHFGPLTWVCKPQGG- (G) 5-F¹</td><td> 340</td><td>EPO-mimetic</td>

<td>GGTYSCHFGPLTWVCKPQGG- (G) 5-F¹</td><td> 1034</td><td>EPO-mimetic</td>

<td>F.¹- (G) s-DFLFIKNTSLGHRP</td><td> 1045</td><td>a TNF-α inhibitor</td>

<td>DFLFIKNTSLGHRP- (G) sF¹</td><td> 1046</td><td>a TNF-α inhibitor</td>

<td>F.¹- (G) s- fewtpgywqpyalpl</td><td> 1047</td><td>an IL-1 R antagonist</td>

<td>FEWTPGYWQPYALPL- (G) sF¹</td><td> 1048</td><td>an IL-1 R antagonist</td>

<td>F.¹- (G) s-VEPNCDIHVMWEWECFERL</td><td> 1049</td><td>a VEGF antagonist</td>

<td>VEPNCDIHVMWEWECFERL- (G) sF¹</td><td> 1050</td><td>a VEGF antagonist</td>

<td>F.¹- (G) s-CTTHWGFTLC</td><td> 1051</td><td>an MMP inhibitor</td>

<td>CTTHWGFTLC- (G) s-F1</td><td> 1052</td><td>an MMP inhibitor</td>

"F¹"Is an Fc domain as previously defined herein

The compounds described above can be prepared as described below. The examples provided include the preferred embodiments of the invention and are illustrative rather than limiting.

Example 1 TPO mimetics

The following example uses peptides identified with the numbers provided in Table A below.

Preparation of Peptide 19. Peptide 17b (12 mg) and MeO-PEG-SH 5000 (30 mg, 2 eq.) Were dissolved in 1 ml of aqueous buffer solution (pH 8). The mixture was incubated at room temperature

For about 30 minutes and the reaction was checked by analytical HPLC which showed that the reaction was over 80% complete. The pegylated material was isolated using a preparative HPLC method.

Preparation of peptide 20. Peptide 18 (14 mg) and MeO-PEG-maleimide (25 mg) were dissolved in approximately 1.5 ml of an aqueous buffer solution (pH 8). The mixture was incubated at room temperature for approximately 30 minutes during which time the transformation was approximately 70% as observed by analytical HPLC by placing a portion of the sample on an HPLC column. The pegylated material was purified by preparative HPLC.

Bioactivity trials. The in vitro TPO bioassay is a mitogenic assay utilizing an IL-3 dependent clone of murine 32D cells that has been transfected with the human mpl receptor. This test is described in detail in WO 95/26746. Cells are maintained in MEM containing 10% Fetal Clone II and 1 ng / ml mIL-3. Before sample addition, cells were prepared by washing twice with mlL-3 free growth medium. An extended TPO standard curve was performed with 12 points ranging from 33 to 39 pg / ml. Four dilutions were made for each sample, selected to fall within the linear portion of the standard curve (100 to 125 pg / ml) and repeated three times. A volume of 100 µl of each sample or standard dilution was added to the appropriate wells of a 96-well microtiter plate containing 10,000 cells / well. After 44 hours at 37 ° C and 10% CO 2, MTS (a tetrazolium compound that is bioreduced by cells to formazan) was added to each well. Approximately six hours later, the optical density was read on the reading plate at 490 nm. A dose / response curve (log TPO concentration versus OD-background) was constructed and linear regression analysis was performed on the points belonging to the linear portion of the standard curve. The concentrations of the unknown samples tested were determined using the obtained linear equation and correction for the dilution factor.

TMP tandem repeats with polyglycine linkers. The design of the sequentially bound TMP dimers was based on the assumption that a dimeric form of TMP is required for its effective interaction with c-Mpl (TPO receptor) and that depending on how they fold together in the receptor context, the two TMP molecules could be bound together in the C-terminus to the N-terminus in a manner which would not disturb the overall dimeric conformation. Clearly, the activity of the tandem-linked dimers may also depend on proper selection of the length selection and composition of the linker that binds the C- and N-terminus of two sequentially adjacent TMP monomers. Since no structural information was available about TMP binding to c-Mpl, a series of dimeric peptide syntheses with linkers consisting of "0" to 10 and 14 glycine residues were performed (Table A). Glycine was chosen for its simplicity and flexibility and for this additional rationale that a flexible polyglycine peptide chain may allow the two linked head-tail TMP repeats to fold freely in the desired conformation, while more spherically hindered amino acid sequences may adopt undesirable secondary structures whose stiffness can disrupt the correct packing of the dimeric peptide in the context of the receptor.

The resulting peptides are readily available by using conventional solid phase peptide synthesis methods (Merrifiled, RB, Journal of the American Chemical Society 85: 2149 (1963)) with either Fmoc or t-Boc chemistry. In contrast to the synthesis of a C-terminally linked parallel dimer (SEQ ID NO: 2), which requires the use of an orthogonally protected lysine residue as the starting branch point for pseudo symmetric generation of the two peptide chains (Cwirla, SE et al., Science 276: 1696) -1699 (1997)), the syntheses of our tandem dimers were a simple, stepwise assembly of continuous peptide chains from C-terminus to N-terminus. Because TMP dimerization has a more dramatic effect on proliferative activity than on binding affinity as shown for the C-terminal dimer. (Cwirla, SE et al., Science 276: 1696-1699 (1997)), synthetic peptides were tested directly for biological activity in a TPO-dependent cell proliferation assay using an IL-3 dependent clone of 32D murine cells transfected with c Full-length MP1 (Palacios, R. et al., Cell 41: 727 (1985)). As shown by the test results (see Table 1 below), all tandem dimers linked with a polyglycine linker showed more than a 1000-fold increase in potency compared to the monomer and even had a higher potency than the C-terminal dimer in the cell proliferation assays. The absolute activity of the C-terminal dimer in our trial was lower than that of the native TPO protein, which differs from the previously reported findings in which the C-terminal dimer was found to be as active as the natural ligand (Cwirla, SE et al. Science 276: 1696-1699 (1997)). This could have happened due to the different conditions used in the two trials.

PL 211 164 B1

Despite this, the differences in activity between tandem dimers (with the C-terminus of the first monomer fused to the N-terminus of the second monomer) and parallel dimers (with the C-terminus of the first monomer fused to the C-terminus of the second monomer) clearly demonstrate tandem superiority in the same trial. dimerized product compared with parallel dimer products. Note that a wide range of lengths is tolerated for the linker. The optimal linker for the selected TMP monomers consists of 8 glycines.

Other tandem repetitions. After the first series of TMP tandem dimers, several other molecules were designed several further molecules either with different linkers or containing modifications within the monomer as such. The first of these molecules, peptide 13, contains a linker consisting of GPNG, a known sequence that is highly prone to forming a β-turn secondary structure. However, with still about 100 times greater potency compared to the monomer, this peptide shows more than 10 times less activity compared to the GGGG linked analogue. Therefore, the introduction of a relatively stiff β-turn in the linker region appears to cause a slight disturbance of the optimal agonist configuration in the short linkers.

Trp9 in the TMP sequence is a highly conserved residue among active peptides selected from random peptide libraries. It is also highly protected Trp in 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 EMO-mimetic peptides and to be involved in hydrophobic interactions with the EPO receptor. Livnah et al. (1996), Science 273: 464-71). By analogy, it was thought that the Trp9 residue in TMP could perform a similar function in peptide ligand dimerization. In an attempt to modulate and evaluate the effects of non-covalent hydrophobic forces exerted by the two indole rings, several analogs were constructed by mutating this Trp. Thus, in peptide 14, the Trp residue was replaced with each of the two TMP monomers for a Cys residue, and an intramolecular disulfide bond was formed between both cysteines by oxidation, which was intended to mimetic mimic the hydrophobic interactions between the two Trp residues in peptide dimerization. Peptide 15 is the reduced form of peptide 14. In peptide 16, two Trp residues were changed to Ala. As shown by the results of the trials, all three analogs were inactive. These results further demonstrated that Trp is important for the activity of the TPO-mimetic peptide, but not only for dimer formation.

The next two peptides (peptides 17a and 18) each contain a Lys or Cys residue in their eight amino acid linker. These two compounds are precursors to two PEGylated peptides (peptides 19 and 20) in which the Lys or Cys side chain is modified by a PEG moiety. It was decided to introduce a PEG moiety in the middle of a relatively long linker such that the large PEG component (5 kDa) was sufficiently distant from the binding sites on the peptide molecule. PEG is a known biocompatible polymer that is increasingly being used as a covalent modifier to improve the pharmacokinetic profiles of peptide and protein based therapeutic agents.

A reference solution-based method was developed for the convenient PEGylation of synthetic or recombinant peptides. The method is based on a well established chemoselective ligation strategy that uses a specific reaction between a pair of mutually reactive functional systems. Thus, to pegylate peptide 19, the lysine side chain was pre-activated with a bromoacetyl group, yielding peptide 17b which was capable of reacting with a PEG thiol derivative. To do this, an orthogonal protecting group, Dde, was included to protect the ε-amino residue in the lysine. When the entire peptide chain was aligned, the N-terminal amine was re-protected using t-Boc. Dde was then removed to allow bromoacetylation. This strategy resulted in a crude peptide that was easily purified using a conventional reverse phase HPLC method. Ligation of the peptide with the PEG-modified thiol group took place in an aqueous buffer at pH 8 and the reaction was complete in 30 minutes. MALDl-MS analysis of the purified pegylated material revealed a characteristic bell-shaped spectrum with an increase of 44 Da between adjacent peaks. In the case of the PEG-peptide 20 system, a cysteine ​​residue was placed in the linker region and its side chain thiol group should serve as the "attachment" site for the maleimide-containing PEG. Similar conditions were used to pegylate this peptide. As revealed by the results of the trials, these two pegylated peptides had even greater in vitro bioactivity than their non-pegylated counterparts.

Peptide 21 has a potential glycosylation motif, NGS, in its eight amino acid linker. Because our sample tandem dimers are made of natural linked amino acids

By peptide bonds, expression of such a molecule in a suitable eukaryotic cell system should result in the production of a glycopeptide with a carbohydrate moiety added to the Asn side chain carboxamide. Glycosylation is a common post-translational modification that can have a very positive effect on the biological activity of a given protein by increasing its water solubility and in vivo stability. As the test results show, the inclusion of this glycosylation motif in the linker maintains high biological activity. The synthetic precursor of the candidate glycopeptide resulted in similar activity to its analog with the linker- (Gly) 8-. Upon glycosylation, this peptide is expected to have the same order of activity as pegylated peptides, due to the similar chemophysical properties exhibited by PEG moieties and carbohydrate moieties.

The last peptide is the tandem repeat dimer. It was produced by the oxidation of peptide 18, which formed an intramolecular disulfide bond between two cysteine ​​residues located on the linker. This peptide was designed to test whether TMP is active as a tetramer. The results of the trial showed that this peptide was not more active than the average tandem dimer per molecule, which indirectly supports the thesis that the active form of TMP is indeed a dimer, otherwise dimerization of the tandem dimer would have an additional effect on biological activity.

To confirm the results obtained in vitro in animal studies, one pegylated tandem TMP "repeat" (compound 20 in Table A) was administered subcutaneously to normal mice using osmotic pumps. Time and dose related increases in platelet count were observed during treatment. Peak platelet levels greater than 4 times baseline were observed on day 8. A dose of 10 µg / kg / day of pegylated TMP "replicate" produced a similar response to rHuMGDF (non-pegylated) at 100 µg / kg / day given by the same route.

Table A - TPO-mimetic peptides

<td>Peptide, No.</td><td>Relationship</td><td>SEQ ID NO:</td><td>Relative power</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>TPO</td><td></td><td></td><td> ++++</td>

<td>TMP monomer</td><td></td><td> 13</td><td> +</td>

<td>TMP CC dimer</td><td></td><td></td><td> +++-</td>

<td>TMP- (G) n-TMP:</td><td></td><td></td><td></td>

<td>1 N = 0</td><td></td><td> 341</td><td> ++++-</td>

<td>2 N = 1</td><td></td><td> 342</td><td> ++++</td>

<td>3 N = 2</td><td></td><td> 343</td><td> ++++</td>

<td>4 N = 3</td><td></td><td> 344</td><td> ++++</td>

<td>5 N = 4</td><td></td><td> 345</td><td> ++++</td>

<td>6 N = 5</td><td></td><td> 346</td><td> ++++</td>

<td>7 N = 6</td><td></td><td> 347</td><td> ++++</td>

<td>8 N = 7</td><td></td><td> 348</td><td> ++++</td>

<td>9 N = 8</td><td></td><td> 349</td><td> ++++-</td>

<td>10 N = 9</td><td></td><td> 350</td><td> ++++</td>

<td>11 N = 10</td><td></td><td> 351</td><td> ++++</td>

<td>12 N = 14</td><td></td><td> 352</td><td> ++++</td>

PL 211 164 B1 cont. table A

<td></td><td> 1 2</td><td> 3</td><td> 4</td>

<td> 13</td><td>TMP-GPNG-TMP</td><td> 353</td><td> +++</td>

<td> 14</td><td>IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA</td><td> 354</td><td> -</td>

<td></td><td>II (cyclic)</td><td></td><td></td>

<td> 15</td><td>IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear)</td><td> 355</td><td> -</td>

<td> 16</td><td>IEGPTLRQALAARA-GGGGGGGG-lEGPTLRQALAARA</td><td> 356</td><td> -</td>

<td>17a</td><td>TMP-GGGKGGGG-TMP</td><td> 357</td><td> ++++</td>

<td>17b</td><td>TMP-GGGK (BrAc) GGGG-TMP</td><td> 358</td><td>undefined</td>

<td> 18</td><td>TMP-GGGCGGGG-TMP</td><td> 359</td><td> ++++</td>

<td> 19</td><td>TMP-GGGK (PEG) GGGG-TMP</td><td> 360</td><td> +++++</td>

<td> 20</td><td>TMP-GGGC (PEG) GGGG-TMP</td><td> 361</td><td> +++++</td>

<td> 21</td><td>TMP-GGGN * GSGG-TMP</td><td> 362</td><td> ++++</td>

<td> 22</td><td>TMP-GGGCGGGG-TMP AND</td><td> 363</td><td> ++++</td>

<td></td><td>TMP-GGGCGGGG-TMP</td><td> 363</td><td></td>

Discussion. It is well established that MGDF acts in a similar way to human growth hormone (hOH), for example, one protein ligand molecule binds two receptor molecules to activate it. Wells et al. (1996), Ann. Rev. Biochem. 65: 609-34. Currently, this interaction mimetic mimics that of a much smaller peptide, TMP. However, current studies suggest that this mimicry phenomenon requires the concerted action of two TMP molecules because covalent TMP dimerization, both parallel CC and sequential increased the in vitro biological potency of the original monomer by a factor greater than 10³. The relatively low biological strength of the monomer is likely due to ineffective non-covalent dimer formation. The pre-formed covalent "repeat" has the ability to eliminate the entropy barrier for dimer formation which is solely caused by weak, non-covalent interactions between two small 14-residue peptide molecules.

It is intriguing that this tandem "repeat" approach has a similar effect in increasing bioactivity as the prior CC dimerization. These two strategies lead to two very different molecular configurations. The CC dimer is a quasi-symmetric molecule, while tandem "repeats" do not have such symmetry in their linear structures. Despite this difference in their primary structures, these two types of molecules proved able to bend efficiently into a similar biologically active conformation and caused c-Mpl dimerization and activation. These experimental observations provide several "insights" on how two TMP molecules can interact when binding to c-Mpl. First, the two C-ends of the two TMP-linked molecules must be in relatively close proximity to each other as suggested by the C-terminal dimer data. Second, the respective N- and C- ends of the two TMP molecules in the receptor complex must be positioned very close to each other so that they can be directly bound together by a single head-tail peptide bond to achieve the effect of an almost maximum increase in activity due to the tandem "repeat" strategy. . Insertion of one or more (up to 14) glycine residues into the junction did not result in any further significant increase (or decrease). This may be due to the fact that the flexible polyglycine peptide chain can easily loop from connection without causing any change in overall conformation. This flexibility seems to imply the introduction of freedom of orientation in the case of TMP peptide chains bending in the required conformation to interact with the receptor and confirms this as a modification site. Indirect evidence for this was obtained from the study of peptide 13, in which the much stiffer b-turn sequence used as a linker apparently forced a deviation from the alignment of the compound backbone around the linker, which may have resulted in a slight disruption of optimal conformation and thus resulted in a mean (10-fold) ) a decrease in activity compared to the analogous compound with a 4-glycine linker. Third, Trp9

The role of TMP in TMP is similar to that of Trp13 in EMP, which is not only affected by the peptide-peptide interaction in dimer formation, but is also an important contributor to the hydrophobic forces in the peptide: receptor interaction. The results obtained for the analogue of the mutant from W to C, peptide 14, suggest that the covalent disulfide linkage is not sufficient to approximate the hydrophobic interactions caused by the Trp pair and that, being a short linkage, it may bring the two TMP monomers too close and therefore introduce perturbations in the overall conformation of the optimal dimeric structure.

Analysis of the possible secondary structure of the TMP peptide may provide further understanding of the interaction between TMP and c-Mpl. This may be facilitated by reference to the described structure of the EPO-mimetic peptide. Livnah et al. (1996), Science 273: 464-75. The EMP-linked receptor has a hairpin b-twist structure formed by the highly compatible Gly-Pro-Leu-Tr system at the center of its sequence.

Instead of GPLT, TMPs have a highly selected GPTL sequence that is similarly prone to forming a similar twist. However, this twist-like pattern is close to the N-terminal part in the TMP. Secondary structure prediction using the Chau-Fasman method suggests that the C-terminal half of the peptide tends to adopt a circular conformation. Together with the highly conserved Trp at position 9, this C-terminal helix can help to stabilize the dimeric structure. It is interesting to note that most of our tandem "repeats" show greater "power" compared to the C-terminal parallel dimer. Tandem "repeats" appear to render the molecule more conformational than the parallel CC dimerization does. Similarly, the asymmetric features of the "repetition" tandem may bring it closer to the natural ligand which, as an asymmetric molecule, uses two different sites to bind two identical receptor molecules.

The introduction of a PEG moiety was predicted to enhance the in vivo activity of the modified peptide by providing protection against proteolytic degradation and by slowing its removal by renal filtration. It was surprisingly found that pegylation could further increase the in vivo bioactivity of the tandem dimerized TMP peptide in a cell proliferation assay.

Example 2

Fc-TMP mergers

TMP (and EMP as described in Example 3) moieties were expressed either in monomeric or dimeric form of either N-terminal or C-terminal fusions to the Fc region of human IgG1. In all cases, the expression construct used the luxPR promoter in the expression vector of the pEMG21 plasmid.

Fc-TMP A DNA sequence encoding a human IgG1 Fc region fused to a TPO-mimetic peptide monomer was constructed using standard PCR technology. The pFc-A3 vector and the synthetic TMP gene were the templates for the PCR reactions. The synthetic gene was constructed from three (3) overlapping oligonucleotides (SEQ ID NOS: 364, 365, and 366, respectively) shown below:

<td> 1842-97</td><td>AAA CTG</td><td>AAA ACG</td><td>GGA CAG</td><td>TCC AGT</td><td>TCG CGG</td><td>AGA ACC</td><td>TTA</td><td>AGC</td><td>ACG</td><td>AGC</td><td>AGC</td><td>CAG</td><td>CCA</td>

<td> 1842-98</td><td>AAA</td><td>GGT</td><td>GGA</td><td>GGT</td><td>GGT</td><td>GGT</td><td>ATC</td><td>GAA</td><td>GGT</td><td>CCG</td><td>ACT</td><td>CTG</td><td>CGT</td>

<td> 1842-99</td><td>CAG TTT</td><td>TGG</td><td>CTG</td><td>GCT</td><td>GCT</td><td>CGT</td><td>GCT</td><td>TAA</td><td>TCT</td><td>CGA</td><td>GGA</td><td>TCC</td><td>TTT</td>

These oligonucleotides were "relaxed" to form a duplex encoding the amino acid sequence (SEQ ID NOS: 367 and 368, respectively), shown below:

AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT 1 ---------₊---------₊---------₊---------+---------₊---------₊ 60

CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA a KGGGGGIEGPTLRQWLAARA TAATCTCGAGGATCCTTTTTT 61 -........ + --------- + - 81

ATTAGAGCTCCTAGGAAAAAA a *

PL 211 164 B1

This duplex was amplified by PCR using 1842-98 and 1842-97 as sense and antisense primers.

The Fc portion of the molecule was generated by PCR with pFc-A3 using the primers shown below (SEQ ID NOS: 369 and 370):

1216-52 AAC ATA AGT ACC TGT AGG ATC G

1830-51 TTCGATACCA CCACCTCCAC CTTTACCCGG AGACAGGGAG AGGCTCTTCTGC

The 1830-51 oligonucleotides contain an overlap of 24 nucleotides leading to the fusion of 2 genes fused together in the correct reading frame by joining the above PCR products in a third reaction using the outer promoters 1216-52 and 1842-97.

The final PCR gene product (full-length fusion gene) was digested with the restriction endonuclease XbaI and BamHl, then ligated into the pAMG21 vector, and transformed into competent E. coli 2596 host cells as described herein for EMP-Fc. Clones were screened for the ability to produce a recombinant protein product and for the presence of a gene fusion with the correct nucleotide sequence. This single clone was selected and designated as strain Amgen # 3728.

The nucleotide and amino acid sequences of the obtained fusion protein (SEQ ID NOS: 5 and 6) are shown in Fig. 7.

Fc-TMP-TMP. A DNA sequence encoding a human IgG1 Fc region fused to a TPO-mimetic peptide dimer was constructed using standard PCR technology. The pFc-A3 vector and the synthetic TMP-TMP gene were the templates for the PCR reaction. A synthetic gene was constructed from the four (4) overlapping oligonucleotides (SEQ ID NOS: 371 to 374 respectively) shown below:

<td> 1830-52</td><td>AAA ACT</td><td>GGT CTG</td><td>GGA CGT</td><td>GGT CAG</td><td>GGT TGG</td><td>GGT CTG</td><td>ATC GCT</td><td>GAA GCT</td><td>GGT CGT</td><td colspan="4">CCG GCT</td>

<td> 1830-53</td><td>ACC</td><td>TCC</td><td>ACC</td><td>ACC</td><td>AGC</td><td>ACG</td><td>AGC</td><td>AGC</td><td>CAG</td><td></td><td></td><td></td><td></td>

<td></td><td>CCA</td><td>CTG</td><td>ACG</td><td>CAG</td><td>AGT</td><td>CGG</td><td>ACC</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1830-54</td><td>GGT</td><td>GGT</td><td>GGA</td><td>GGT</td><td>GGC</td><td>GGC</td><td>GGA</td><td>GGT</td><td>ATT</td><td>GAG</td><td>GGC</td><td>CCA</td><td>ACC</td>

<td></td><td>CTT</td><td>CGC</td><td>CAA</td><td>TGG</td><td>CTT</td><td>GCA</td><td>GCA</td><td>CGC</td><td>GCA</td><td></td><td></td><td></td><td></td>

<td> 1830-55</td><td>AAA</td><td>AAA</td><td>AGG</td><td>ATC</td><td>CTC</td><td>GAG</td><td>ATT</td><td>ATG</td><td>CGC</td><td>GTG</td><td>CTG</td><td>CAA</td><td>GCC</td>

<td></td><td>ATT</td><td>GGC</td><td>GAA</td><td>GGG</td><td>TTG</td><td>GGC</td><td>CCT</td><td>CAA</td><td>TAC</td><td>CTC</td><td>CGC</td><td>CGC</td><td>C.</td>

These 4 oligonucleotides were "relaxed" to form a duplex encoding the amino acid sequence (SEQ ID NOS: 375 and 376, respectively) shown below:

AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT 1 --------- + --------- + --------- + ---------₊---------₊--------- + it

CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA a KGGGGGIEGPTLRQWLAARA GGTGGTGGAGGTGGCGGCGGAGGTATTGAGGGCCCAACCCTTCGCCAATGGCTTGCAGCA

---------- 1 ----------- H ---------- 1 ----------- 1 ---- ------- 1 ----------- p 12 0

CCACCACCTCCACCGCCGCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGT a GGGGGGGGIEGPTLRQWLAA CGCGCA

121 ----------------------------------148

GCGCGTATTAGAGCTCCTAGGAAAAAAA a RA * This duplex was PCR amplified using 1830-52 and 1830-55 as sense and antisense primers.

PL 211 164 B1

The FC portion of the molecule was generated by PCR with pFC-A3 using primers 1215-52 and

1830-51 as described above for Fc-TMP. A full-length fusion gene was obtained from the third reaction

PCR using external primers 1215-52 and 1830-55.

The final PCR gene product (full-length fusion gene) was digested with the restriction endonuclease XbaI and BamHl, then ligated into the pAMG21 vector, and transformed into competent E. coli 2595 host cells as described in Example 1. Clones were screened for the ability to produce recombinant protein product and having the fusion gene with the correct nucleotide sequence. This single clone was selected and designated as strain # 3727 from Amgen.

The nucleotide and amino acid sequences of the fusion protein (SEQ ID NOS: 7 and 8) are shown in Figure 8.

TMP-TMP-Fc. A DNA sequence encoding a tandem "repeat" of the TPO-mimetic peptide fused to the Fc region of human IgG1 was constructed using standard PCR technology. The templates for the PCR reaction were the EMP-Fc plasmid from strain # 3588 (see example 3) and the synthetic gene encoding the TMP dimer. The synthetic gene for the tandem "repeat" was constructed from the seven (7) overlapping oligonucleotides shown below (SEQ ID NOS: 377 to 383, respectively):

<td> 1885-52</td><td>TTT</td><td>TTT</td><td>CAT</td><td>ATG</td><td>ATC</td><td>GAA</td><td>GGT</td><td>CCG</td><td>ACT</td><td>CTG</td><td>CGT</td><td>CAG</td><td>TGG</td>

<td> 1885-53</td><td>AGC GAT</td><td>ACG CAT</td><td>AGC ATG</td><td>AGC</td><td>CAG</td><td>CCA</td><td>CTG</td><td>ACG</td><td>CAG</td><td>AGT</td><td>CGG</td><td>ACC</td><td>TTC</td>

<td> 1885-54</td><td>CTG CAC</td><td>GCT ACA</td><td>GCT</td><td>CGT</td><td>GCT</td><td>GGT</td><td>GGA</td><td>GGC</td><td>GGT</td><td>GGG</td><td>GAC</td><td>AAA</td><td>ACT</td>

<td> 1885-55</td><td>CTG ATT</td><td>GCT GAG</td><td>GCT GGC</td><td>CGT CCA</td><td>GCT</td><td>GGC</td><td>GGT</td><td>GGT</td><td>GGC</td><td>GGA</td><td>GGG</td><td>GGT</td><td>GGC</td>

<td> 1885-56</td><td>AAG TCC</td><td>CCA GCC</td><td>TTG ACC</td><td>GCG ACC</td><td>AAG GCC</td><td>GGT</td><td>TGG</td><td>GCC</td><td>CTC</td><td>AAT</td><td>GCC</td><td>ACC</td><td>CCC</td>

<td> 1885-57</td><td>ACC GGT</td><td>CTT GGG</td><td>CGC GAC</td><td>CAA AAA</td><td>TGG ACT</td><td>CTT</td><td>GCA</td><td>GCA</td><td>CGC</td><td>GCA</td><td>GGG</td><td>GGA</td><td>GGC</td>

<td> 1885-58</td><td>CCC</td><td>ACC</td><td>GCC</td><td>TCC</td><td>CCC</td><td>TGC</td><td>GCG</td><td>TGC</td><td>TGC</td><td></td><td></td><td></td><td></td>

These oligonucleotides were "relaxed" to form a duplex encoding the amino acid sequence shown below (SEQ ID NOS: 384 and 385):

TTTTTTCATATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCTGGCGGT 1 --------- + ---------₊---------₊---------₊---------+---------+ _eo

GTATACTAGCTTCCAGGCTGAGACGCAGTCACCGACCGACGAGCACGACCGCCA MIEGPTLRQWLAARAGG GGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCAATGGCTGGCTGCTC ---------₊---------₊---------₊--------- + --------- + --------- + i2o

CCACCGCCTCCCCCACCGTAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGTGCGCGT a GGGGGGIEGPTLRQWLAARA GGTGGAGGCGGTGGGGACAAAACTCTGGCTGCTCGTTGA- + ---- + --------- + 180

CCCCCTCCGCCACCC a GGGGGDKTLAARAGGGGGDK ACTCACACA 181 --------- 189 a Τ Η T PL 211 164 B1

This duplex was amplified by PCR using 1885-52 and 1885-58 as sense and antisense primers.

The FC portion of the molecule was generated by PCR with DNA from the EMP-Fc fusion of strain # 3688 (see example 3) using primers 1885-54 and 1200-54. A full-length fusion gene was obtained from the third PCR reaction using the external primers 1885-52 and 1200-54.

The final PCR gene product (full-length fusion gene) was digested with restriction endonuclease XbaI and BamHI, then ligated into the pAMG21 vector and transformed into competent E. coli 2596 host cells as described for Fc-EMP. Clones were screened for the ability to produce a recombinant protein product and for having a gene fusion with the correct nucleotide sequence. This single clone was selected and designated as strain # 3798 from Amgen.

The nucleotide and amino acid sequences of the fusion protein (SEQ ID NOS: 9 and 10) are shown in Figure 9.

TMP-Fc. Fortunately, during the ligation into TMP-TMP-Fc, a DNA sequence encoding a TPO-mimetic peptide monomer fused in frame to the Fc region of human IgG1, presumably due to the ability of the 1885-54 primer to "relax" to 1885-53, as well as to 1885-58. A single clone with the correct nucleotide sequence for the TMP-Fc construct was selected and designated as Amgen strain # 3788.

The nucleotide and amino acid sequences of the fusion protein (SEQ ID NOS: 11 and 12) are shown in Figure 10.

Expression in E. coli. Cultures of all pAMG21-Fc-TMP-TMP fusion construct in E. coli GM221 in Luria Broth medium containing 50 g / ml kanamycin were incubated at 37 ° C. The induction of gene product expression from the luxPR promoter was achieved by adding the synthetic N- (3-oxohexanoyl) -DL-homoserine lactone auto-inducer to the culture medium to a final concentration of 20 ng / ml and the cultures were incubated at 37 ° C for a further 3 hours. After 3 hours, the bacterial cultures were examined under a microscope for the presence of inclusion bodies and then harvested by centrifugation. In the induced cultures, refractive inclusion bodies were observed showing that Fc-fusions were most likely produced in the insoluble fraction in E. coli. Cell plates were directly lysed by resuspension in Laemmli sample buffer containing 10% β-mercaptoethanol and analyzed by SDS-PAGE. In each case, an intensely stained Coomassie band of approximately 30 kDa was observed on the SDS-PAGE gel.

pAMG21. The pAMG21 expression plasmid can be derived from the Amgen expression vector pCFM1656 (ATCC # 69576), which in turn is derived from the Amgen expression vector system described in US Patent No. 4,710,473. The plasmid pCFM1656 can be derived from the described plasmid pCFM836 (Patent No. 4,710,473) by:

(a) destroying the two endogenous NdeI restriction sites by filling the ends of the T4 polymerase enzyme followed by ligation of the blunt ends;

(b) replacing the DNA sequence between the unique restriction sites of AatII and Clal containing the synthetic P1 promoter with a similar fragment obtained from pCFM636 (Patent No. 4,710,473), including the PL promoter (see SEQ ID nO: 386 below); and (c) replacing the small DNA sequence between the unique Clal and KpnI restriction sites with an olynucleotide of SEQ ID NO: 388.

SEQ ID NO: 386:

Aatll

5 'CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT3' TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA -AAAAAACATACAGTGATGATTATACAGTGATGATTA

-TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTTTACCACTGGCGGTGATACTGAGCACAT 3 '-ATGGTGACCGCCACTATGACTCGTGTAGC 5'

Clal

PL 211 164 B1

SEQ ID NO: 387:

5 'CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3'

3 'TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5'

Bodies of KpnI

Thus, the pAMG21 expression plasmid can be derived from pCFM1656 by performing a series of site-directed base changes by oligo mutagenesis involving PCR and DNA sequence substitutions. Starting from the BglII site (bp # 180 plasmid) directly 5 'to the pcopb plasmid replication promoter and proceeding towards the plasmid replication genes, the base pair changes are as shown in Table B below.

Table B - Base pair changes leading to pAMG21

<td>pAMG21 bp #</td><td>bp wpCFM1656</td><td>changed bp in pAMG21</td>

<td> # 204</td><td>T / A</td><td>C / G</td>

<td> # 428</td><td>A / T</td><td>G / C</td>

<td> # 509</td><td>G / C</td><td>A / T</td>

<td> # 617</td><td> - -</td><td>insertion of two G / C bp</td>

<td> # 679</td><td>G / C</td><td>T / A</td>

<td> # 980</td><td>T / A</td><td>C / G</td>

<td> # 994</td><td>G / C</td><td>A / T</td>

<td> # 1004</td><td>A / T</td><td>C / G</td>

<td> # 1007</td><td>C / G</td><td>T / A</td>

<td> # 1028</td><td>A / T</td><td>T / A</td>

<td> # 1047</td><td>C / G</td><td>T / A</td>

<td> # 1178</td><td>G / C</td><td>T / A</td>

<td> # 1466</td><td>G / C</td><td>T / A</td>

<td> # 2028</td><td>G / C</td><td>base pair deletion</td>

<td> # 2187</td><td>C / G</td><td>T / A</td>

<td> # 2480</td><td>A / T</td><td>T / A</td>

<td> # 2499-2502</td><td>AGTG TCAC</td><td>GTCA CAGT</td>

<td> # 2642</td><td>TCCGAGC AGGCTCG</td><td>7 base pair deletion</td>

<td> # 3435</td><td>G / C</td><td>A / T</td>

<td> # 3446</td><td>G / C</td><td>A / T</td>

<td> # 3643</td><td>A / T</td><td>T / A</td>

The DNA sequence between the unique restriction sites AatII (position # 4364 in pCFM1656) and SacII (position # 4585 in pCFM1656) is substituted with the DNA sequence (SEQ ID NO: 23) shown in Figures 17A and 17B. During ligation of the sticky ends of this replacement DNA sequence, the outer AatII and SacII sites were damaged. These are unique AatII and SacII sites in the substituted DNA.

GM221 (Amgen # 2596). Amgen's # 2596 host strain is E. coli K-12 strain, which has been modified to contain both the temperature-sensitive cI857s7 lambda repressor in the early ebg region and the lacI repressor^Q in the later ebg area (68 minutes). The presence of these two repressor genes allows this host to be used with different expression systems, although both of these repressors are unsuitable for expression using luxPR. The non-transformed host is not resistant to antibiotics.

PL 211 164 B1

The ribosomal binding site of the cI857s7 gene was modified to include a boosted one

RBS. It was inserted into the ebg operon between nucleotide positions 1170 and 1411 according to Genbank (deposit) numbering under accession number M64441Gb_Ba, with the deletion of the interfering ebg sequence. The sequence of the insert is shown below, with lowercase fonts being used to write the ebg sequence on the flanks of the insert (SEQ ID NO: 388) shown below:

ttattttcgtGCGGCCGCACCATTATCACCGCCAGAGGTAAACTAGTCAACACGCACGGTGTTAGATATTTATCCC TTGCGGTGATAGATTGAGCACATCGATTTGATTCTAGAAGGAGGGATAATATATGAGCACAAAAAAGAAACCATTA ACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTGGCTTATCCC AGGAATCTGTCGCAGACAAGATGGGGATGGGGCAGTCAGGCGTTGGTGCTTTATTTAATGGCATCAATGCATTAAA TGCTTATAACGCCGCATTGCTTACAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCCTTCAATCGCCAGAGAA TCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTACCCTGTTTTTTCTCATGT TCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTACCAAAGGTGATGCGGAGAGATGGGTAAGCACAACCAAA AAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCGCACCAACAGGCTCCAAGCCAAGCT TTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCCAGGTGATTTCTGCATAGCCAGACTTGG GGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAGGTGTTTTTACAACCACTAAACCCACAGTAC CCAATGATCCCATGCAATGAGAGTTGTTCCGTTGTGGGGAAAGTTATCGCTAGTCAGTGGCCTGAAGAGACGTTTG GCTGATAGACTAGTGGATCCACTAGTg tttctgccc

The construct was delivered to the chromosome using a recombinant phage named MMebgcI857s7 enhanced with RBS # 4 in F'tet / 393. After recombination and separation, only the chromosomal insert described above remains in the cell. It was renamed F'tet / GM101. The F'tet / GM101 was then modified to provide the lacI construct^Q to operon ebg between nucleotide positions 2493 and 2937, according to Genbank (deposit) numbering under accession number M64441Gb_Ba, with deletion of the disturbing sequence ebg. The sequence of the insert is shown below, with the lowercase font used to write the ebg sequence on the flanks of the insert (SEQ ID NO: 389) shown below:

ggcggaaaccGACGTCCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATT

CAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCC

CGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTCGAAGCGGCGATGGCGGAGCTGAATT

ACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGCTCCTGATTGGCGTTGCCACCTCCAGTCTGGC

CCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCG

ATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGA

TCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCT

TGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCAT

CTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGG

CTGGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTC

CGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATG

GCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACG

ATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAG

CGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAA

AGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCAC

GACAGGTTTCCCGACTGGAAAGCGGACAGTAAGGTACCATAGGATCCaggcacagga

The construct was delivered to the chromosome using a recombinant phage named AGebgLacIQ # 5 in F'tet / GM101. After recombination and separation, only the chromosomal insert described above remains in the cell. It was renamed F'tet / GM221. Episom F'tet was separated from the strain using acridine orange at a concentration of 25 µg / ml in LB. The separated strain was identified as tetracycline sensitive and stored as GM221.

Expression. Cultures of pAMG21-Fc-TMP-TMP in E. coli GM221 in Luria Broth medium containing 50 µg / ml kanamycin were incubated at 37 ° C before induction. The induction of expression of the Fc-TMP-TMP gene product from the luxPR promoter was achieved by adding the synthetic auto-inducer N- (3-oxohexanoyl) -DL-homoserine lactone to the culture medium to a final concentration of 20 ng / ml and the cultures were incubated at 37 ° C for a further 3 hours. After 3 hours, the bacterial cultures were examined under a microscope for the presence of inclusion bodies and then harvested by centrifugation. Refractive inclusion bodies were observed in the induced cultures showing that Fc-TMP-TMP was most likely produced in an insoluble fraction in E. coli. Cell plates were directly lysed by resuspension in Laemmli sample buffer containing 10% β-mercaptoethanol and analyzed by SDS-PAGE. Inten74 was observed

A vivid Coomassie stained band of approximately 30 kDa on an SDS-PAGE gel. The expected gene product should be 269 amino acids in length and have an expected molecular weight of approximately 29.5 kDa. Fermentation was also performed under standard batch conditions on a 10 liter scale, obtaining similar Fc-TMP-TMP expression levels to those obtained on a laboratory scale.

Purification of Fc-TMP-TMP. The cells were disrupted in water (1/10) by the elevated pressure homogenization method (2 runs at 14,000 PSI = approx. 1.0 x 10⁸ Pa) and the inclusion bodies were collected by centrifugation (4,200 rpm in a J-6B for 1 hour). Inclusion bodies were solubilized in 6M guanidine, 50mM Tris, 8mM DTT, pH 8.7 for 1 hour in a 1/10 ratio. The solubilized mixture was diluted 20-fold in 2 M urea, 50 mM Tris, 160 mM arginine, 3 mM cysteine, pH 8.5. The mixture was stirred overnight under cool conditions. At this point in the procedure, the Fc-TMP-TMP monomer subunit dimerizes to form a disulfide-linked compound with the structure shown in Figure 6C. The (product) was then concentrated approximately 10 times by ultrafiltration. It was then diluted 3-fold with 10 mM Tris, 1.5 M urea, pH 9. The pH of this mixture was then adjusted to pH 5 with acetic acid. The pellet was removed by centrifugation and the supernatant was placed on a SP-Sepharose Fast Flow column equilibrated in 20 mM NaAc, 100 mM NaCl, pH 5 (10 mg / ml protein loading, room temperature). The protein was eluted with a 20-column volume of the same buffer with a concentration gradient from 100 mM NaCl to 500 mM NaCl. The column stock was diluted 3-fold and placed on a SP-Sepharose HP column in 20 mM NaAc, 150 mM NaCl, pH 5 (10 mg / ml protein loading, room temperature). The protein was eluted with a 20-column volume of the same buffer with a concentration gradient from 150 mM NaCl to 400 mM NaCl. The peak harvest was collected and filtered.

Characterization of Fc-TMP activity. The in vivo mouse data for the various compounds of the present invention are summarized below.

Mice. Normal female BDF1 at approximately 10-12 weeks of age.

Method of exsanguination: Ten mice in the treatment group on day "0", two groups of 20 mice per group, began the trials 4 days apart. Five mice were bled at each time point, the mice were bled a minimum of three times a week. Mice were anesthetized with isoflurane and a total blood volume of 140-160 µl was obtained by puncturing the ocular sinus. Blood was counted using the mouse blood program for the Technicon H1E blood analyzer. The following parameters were measured: white blood cells, red blood cells, hematocrit, hemoglobin, platelets and neutrophils.

Dosing: Mice were either subcutaneously injected with a single bolus dose or 7-day microosmotic pumps were implanted for continuous delivery of agent. Subcutaneous injections were given in a volume of 0.2 ml. Osmotic pumps were inserted into subcutaneous incisions made in the skin between the shoulder blades while dormant. Compounds were diluted in PBS with 0.1% BSA. All experiments were conducted with one control group, designated by the term "vehicle", to which only the above diluent was administered. The concentration of the test materials in the pumps was adjusted so that the calibrated outflow produced the treatment levels indicated in the accompanying graphs.

Compounds: Titrated doses of compound were administered to mice using a 7-day micro-osmotic pump. Mice were dosed with the various compounds as a single dose of 100 µg / kg in a 7-day micro-osmotic pump. Several of the same compounds were then injected into mice as a single bolus dose.

Activity test results. The results of the activity experiments are shown in Figures 11 and 12. In the dose-dependency studies using 7-day micro-osmotic pumps, the maximum effect was seen with the compound with SEQ ID NO: 18 at 100 μg / kg / day. ; the dose of 10 µg / kg / day was approximately 50% of the maximum activity, and the dose of 1 µg / kg / day was the lowest dose for which activity could be observed in this trial. The activity of the compound at a dose of 10 µg / kg / day was almost equal to that of a dose of 100 µg / kg / day of non-pegylated rHu-MGDF in the same experiment.

Example 3

Fc-EMP mergers

Fc-EMP A DNA sequence encoding a human IgG1 Fc region fused to an EPO-mimetic peptide monomer was constructed using standard PCR technology. The templates for the PCR reaction were a vector containing the Fc sequence (pFc-A3 described in International Application No. WO 97/23614, published July 3, 1997) and a synthetic gene encoding EMP. Synthetic

The monomer gene was constructed from four (4) overlapping oligonucleotides (respectively

SEQ ID NOS: 390 to 392) shown below:

<td> 1798-2</td><td>TAT CCA</td><td>GAA CTT</td><td>AGG CGG</td><td>TGG CCC</td><td>AGG GCT</td><td>TGG GAC</td><td>TGG TTG</td><td>TGG G.</td><td>AGG</td><td>TAC</td><td>TTA</td><td>CTC</td><td>TTG</td>

<td> 1798-3</td><td>CGG</td><td>TTT</td><td>GCA</td><td>AAC</td><td>CCA</td><td>AGT</td><td>CAG</td><td>CGG</td><td>GCC</td><td>GAA</td><td>GTG</td><td>GCA</td><td>AGA</td>

<td></td><td>GTA</td><td>AGT</td><td>ACC</td><td>TCC</td><td>ACC</td><td>ACC</td><td>ACC</td><td>TCC</td><td>ACC</td><td>TTT</td><td>CAT</td><td></td><td></td>

<td> 1798-4</td><td>GTT</td><td>TGC</td><td>AAA</td><td>CCG</td><td>CAG</td><td>GGT</td><td>GGC</td><td>GGC</td><td>GGC</td><td>GGC</td><td>GGC</td><td>GGT</td><td>GGT</td>

<td></td><td>ACC</td><td>TAT</td><td>TCC</td><td>TGT</td><td>CAT</td><td>TTT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1798-5</td><td>CCA</td><td>GGT</td><td>CAG</td><td>CGG</td><td>GCC</td><td>AAA</td><td>ATG</td><td>ACA</td><td>GGA</td><td>ATA</td><td>GGT</td><td>ACC</td><td>ACC</td>

<td></td><td>GCC</td><td>GCC</td><td>GCC</td><td>GCC</td><td>GCC</td><td>ACC</td><td>CTG</td><td></td><td></td><td></td><td></td><td></td><td></td>

These 4 oligonucleotides were "relaxed" to form a duplex encoding the amino acid sequence (SEQ ID NOS: 394 and 395, respectively), shown below:

TATGAAAGGTGGAGGTGGTGGTGGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTG

---------- f .--------- + ---------- f ---------- + ------ --- + --------- + 6 0

TACTTTCCACCTCCACCACCACCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAAC b MKGGGGGGGTYSCHFGPLTWGGTTTGCAAACCGCAGGGTGGCGGCGGCGGCGGCGGTGGTAC ----------- + ---- --- - + ---------- + ---------- + - 133

CCAAACGTTTGGCGTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTGGACC b VCKPQGGGGGGGGTYSCHF This duplex was amplified by PCR using

1798-18

GCA GAA GAG CCT CTC CCT GTC TCC GGG TAA AGG TGG AGG TGG TGG TGG AGG TAC TTA CTCT and

1798-19 CTA ATT GGA TCC ACG AGA TTA ACC ACC CTG CGG TTT GCA A as sense and antisense primers (SEQ ID NOS: 396 and 397, respectively). The Fc part of the molecule was made by PCR with pFc-A3 using primers:

<td> 1216-52</td><td>AAC</td><td>ATA</td><td>AGT</td><td>ACC</td><td>TGT</td><td>AGG</td><td>ATC</td><td>G.</td>

<td> 1798-17</td><td>AGA</td><td>GTA</td><td>AGT</td><td>ACC</td><td>TCC</td><td>ACC</td><td>ACC</td><td>ACC TCC ACC TTT ACC CGG</td>

<td></td><td>AGA</td><td>CAG</td><td>GGA</td><td>GAG</td><td>GCT</td><td>CTT</td><td>CTG</td><td>C.</td>

which are, respectively, SEQ ID NOS: 398 and 399. Oligonucleotides 1798-17 and 1798-18 contain an overlapping 61 nucleotides which allows both genes to be fused in the correct reading frame by combining the above PCR products in a third reaction using the external primers, 1216 -52 and 1798-19.

The final PCR gene product (full-length fusion gene) was digested with restriction endonuclease XbaI and BamHI and ligated into the pAMG21 vector (described below), also digested with Xbal and BamHI. The ligated DNA was transformed into competent host cells of the E. coli 2596 strain (GM221, described herein). Clones were screened for the ability to produce a recombinant protein product and for having a gene fusion with the correct nucleotide sequence. This single clone was selected and designated as strain Amgen # 3718

The nucleotide and amino acid sequences of the obtained fusion protein (SEQ ID NOS: 15 and 16) are shown in Fig. 13.

EMP-Fc. A DNA sequence encoding an EPO-mimetic peptide monomer fused in frame to the Fc region of human IgG1 was constructed using standard PCR technology. The pFC-A3a vector and the synthetic gene encoding the EPO monomer were the templates for the PCR reaction. A synthetic monomer gene was constructed from 4 overlapping oligonucleotides 1798-4 and 1798-5

PL 211 164 B1 (above) and 1798-6 and 1798-7 (SEQ ID NOS: 400 and 401, respectively) shown below:

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

4 oligonucleotides were relaxed to form a duplex encoding the amino acid sequence (SEQ ID NOS: 402 and 403, respectively) shown below:

GTTTGCAAACCGCAGGGTGGCGGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGC

GTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCG A VCKPQGGGGGGGGTYSCHFG.

CCGCTGACCTGGGTATGTAAGCCACAAGGGGGTGGGGGAGGCGGGGGGTAATCTCGAG '

---------4- ---------4-------—--+---------4----------+---------4-- 122

GGCGACTGGACCCATACATTCGGTGTTCCCCCACCCCCTCCGCCCCCCATTAGAGCTCCTAG A PLTWVCKPQGGGGGGG *

This duplex was amplified by PCR using 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 CCC T

GCC TCC CCC ACC as sense and antisense primers (SEQ ID NOS: 404 and 405, respectively). Part of the Fc molecule was made by PCR with pFc-A3 using primers

1798-23 AGG GGG TGG GGG AGG CGG GGG GGA CAA AAC TCA CAC ATG TCC A and

00-54 GTT ATT GCT CAG CGG TGG CA which are, respectively, SEQ ID NOS: 406 and 407. Oligonucleotides 1798-22 and 1798-23 contain 43 overlapping nucleotides, which allows the fusion of both genes in the correct reading frame by joining of the above PCR products in a third reaction using the external primers, 1787-21 and 1200-54.

The final PCR gene product (full-length fusion gene) was digested with the restriction endonuclease XbaI and BamHI, then ligated into the pAMG21 vector and transformed into competent E. coli 2596 host cells as described above. Clones were screened for the ability to produce a recombinant protein product and for the presence of a gene fusion with the correct nucleotide sequence. This single clone was selected and designated as strain Amgen # 3688.

The nucleotide and amino acid sequences of the obtained fusion protein (SEQ ID NOS: 17 and 18) are shown in Fig. 14.

EMP-EMP-Fc. A DNA sequence encoding an EPO-mimetic peptide dimer fused in frame to the Fc region of human IgG1 was constructed using standard PCR technology. The templates for the PCR reaction were the above EMP-Fc plasmid from strain # 3688 and the synthetic gene encoding the EPO dimer. A synthetic gene for the dimer was constructed from 8 overlapping oligonucleotides (SEQ ID NOS: 408 to 415, respectively) shown below:

<td> 1869-23</td><td>TTT TAG</td><td>TTT AAG</td><td>ATC GAG</td><td>GAT GAA</td><td>TTG TAA</td><td>ATT AAT</td><td>CTA ATG</td><td>GAT</td><td>TTG</td><td>AGT</td><td>TTT</td><td>AAC</td><td>TTT</td>

<td> 1869-48</td><td>TAA AA</td><td>AAG</td><td>TTA</td><td>AAA</td><td>CTC</td><td>AAA</td><td>TCT</td><td>AGA</td><td>ATC</td><td>AAA</td><td>TCG</td><td>ATA</td><td>AAA</td>

PL 211 164 B1

<td> 1871-72</td><td>GGA GTT</td><td>GGT TGC</td><td>ACT AAA</td><td>TAC CCG</td><td>TCT</td><td>TGC</td><td>CAC</td><td>TTC</td><td>GGC</td><td>CCG</td><td>CTG</td><td>ACT</td><td>TGG</td>

<td> 1871-73</td><td>AGT</td><td>CAG</td><td>CGG</td><td>GCC</td><td>GAA</td><td>GTG</td><td>GCA</td><td>AGA</td><td>GTA</td><td>AGT</td><td>ACC</td><td>TCC</td><td>CAT</td>

<td></td><td>ATT</td><td>TTA</td><td>TTC</td><td>CTC</td><td>CTT</td><td>C.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1871-74</td><td>CAG</td><td>GGT</td><td>GGC</td><td>GGC</td><td>GGC</td><td>GGC</td><td>GGC</td><td>GGT</td><td>GGT</td><td>ACC</td><td>TAT</td><td>TCC</td><td>TGT</td>

<td></td><td>CAT</td><td>TTT</td><td>GGC</td><td>CCG</td><td>CTG</td><td>ACC</td><td>TGG</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1871-75</td><td>AAA</td><td>ATG</td><td>ACA</td><td>GGA</td><td>ATA</td><td>GGT</td><td>ACC</td><td>ACC</td><td>GCC</td><td>GCC</td><td>GCC</td><td>GCC</td><td>GCC</td>

<td></td><td>ACC</td><td>CTG</td><td>CGG</td><td>TTT</td><td>GCA</td><td>AAC</td><td>CCA</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1871-78</td><td>GTA</td><td>TGT</td><td>AAG</td><td>CCA</td><td>CAA</td><td>GGG</td><td>GGT</td><td>GGG</td><td>GGA</td><td>GGC</td><td>GGG</td><td>GGG</td><td>GAC</td>

<td></td><td>AAA</td><td>ACT</td><td>CAC</td><td>ACA</td><td>TGT</td><td>CCA</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 1871-79</td><td>AGT</td><td>TTT</td><td>GTC</td><td>CCC</td><td>CCC</td><td>GCC</td><td>TCC</td><td>CCC</td><td>ACC</td><td>CCC</td><td>TTG</td><td>TGG</td><td>CTT</td>

<td></td><td>ACA</td><td>TAC</td><td>CCA</td><td>GGT</td><td>CAG</td><td>CGG</td><td>GCC</td><td></td><td></td><td></td><td></td><td></td><td></td>

8 oligonucleotides were relaxed to form a duplex encoding the amino acid sequence (SEQ ID NOS: 416 and 417, respectively) shown below:

_{TTTTTTATCGATTTGATTCTAGATTTGAGTTTTAACTTTTAGAAGGAGGAATAAAATATG}

---------₊---------₊---------₊---------₊---------₊---------₊ 6 O

AAAAAATAGCTAAACTAAGATCTAAACTCAAAATTGAAAATCTTCCTCCTTATTTTATAC a M GGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTGGGTTTGCAAACCGCAGGGTGGC

------ + --------- h ----- - - - - + --------- + --------- + - - - - - (- 120

CCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAACCCAAACGTTTGGCGTCCCACCG a GGTYSCHFGPLTWVCKPQGG GGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGACCTAG - + ------- + 121 ---- + 121 ---- --- + - ------ + 180

CCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTGGACCCATACATTC a GGGGGGTYSCHFGPLTWVCK CCACAAGGGGGTGGGGGAGGCGGGGGGGACAAAACTCACACATGTCCA--1 ---------------- + ---- +----------------1 ---- --- 228

GGTGTTCCCCCACCCCCTCCGCCCCCCCTGTTTTGA a PQGGGGGGGDKTHTCP This duplex was PCR amplified using 1869-23 and 1871-79 (shown above) as sense and antisense primers.

The Fc portion of the molecule was generated by PCR with the DNA strain 3688 using primers 1798-23 and 1200-54 (shown above).

The 1871-79 and 1798-23 oligonucleotides contain 31 overlapping nucleotides, allowing both genes to be fused in the correct reading frame by combining the above PCR products in a third reaction using the outer primers, 1869-23 and 1200-54.

The final PCR gene product (full-length fusion gene) was digested with restriction endonuclease XbaI and BamHI, then ligated into the pAMG21 vector, and transformed into competent E. coli 2596 host cells as described for Fc-EMP. Clones were screened for the ability to produce a recombinant protein product and for having a gene fusion with the correct nucleotide sequence. This single clone was selected and designated as strain Amgen # 3813.

The nucleotide and amino acid sequences of the resulting fusion protein (SEQ ID NOS: 19 and 20, respectively) are shown in Figure 15. This is a "silent" mutation at position 145 (A to G, in bold) such that the final construct has a different sequence nucleotide than the oligonucleotide 1871-72 of which it is derived.

Fc-EMP-EMP. A DNA sequence encoding an EPO-mimetic peptide dimer fused in frame to the Fc region of human IgG1 was constructed using standard PCR technology. The templates for the PCR reaction were plasmids from strains # 3688 and 3813, above.

PL 211 164 B1

The Fc portion of the molecule was generated by PCR with the DNA strain 3688 using primers

1216-52 and 1798-17 (shown above). The part of the molecule containing the EMP dimer was the product of a second PCR with strain DNA 3813, using primers 1798-18 (also shown above) and SEQ ID NO: 418 shown below:

1798-20 CTA ATT GGA TCC TCG AGA TTA ACC CCC TTG TGG CTT ACAT

The 1798-17 and 1798-18 oligonucleotides contain an overlapping 61 nucleotides allowing both genes to be fused in the correct reading frame by combining the above PCR products in a third reaction using the outer primers, 1216-52 and 1798-20.

The final PCR gene product (full-length fusion gene) was digested with restriction endonuclease XbaI and BamHI, then ligated into the pAMG21 vector and transformed into competent E. coli 2596 host cells as described for Fc-EMP. Clones were screened for the ability to produce a recombinant protein product and for the presence of a gene fusion with the correct nucleotide sequence. This single clone was selected and designated as strain Amgen # 3822.

The nucleotide and amino acid sequences (SEQ ID NOS: _ and _, respectively) of the fusion protein are shown in Figure 16.

Characterization of Fc-EMP activity. The study was conducted in vivo as follows.

Mice. Normal female BDF1 at approximately 10-12 weeks of age.

Method of exsanguination: Ten mice in the treatment group on day "0", two groups of 20 mice per group, began the trials 4 days apart. Five mice were bled at each time point, the mice were bled a minimum of three times a week. Mice were anesthetized with isoflurane and a total blood volume of 140-160 µl was obtained by puncturing the ocular sinus. Blood was counted using the mouse blood program for the Technicon H1E blood analyzer. The following parameters were measured: WBC white blood cells, RBC red blood cells, HCT hematocrit, HGB hemoglobin, PLT platelets, NEUT neutrophils, and LYMPH.

Dosing: Mice were either subcutaneously injected with a single bolus dose or 7-day micro-osmotic pumps were implanted for continuous delivery of agent. Subcutaneous injections were given in a volume of 0.2 ml. Osmotic pumps were inserted into subcutaneous incisions made in the skin between the shoulder blades while dormant. Compounds were diluted in PBS with 0.1% BSA. All experiments were conducted with one control group, designated by the term "vehicle", to which only the above diluent was administered. The concentration of the test materials in the pumps was adjusted so that the calibrated outflow produced the treatment levels indicated in the accompanying graphs.

Experiments: Various Fc-conjugated EPO-mimetic peptides (EMPs) were injected into mice as one bolus dose of 100 µg / kg. Fc-EMP fusions were administered to mice using 7-day microosmotic pumps. The pushups were not replaced after 7 days. Mice were bled until day 51 when HGB and HCT returned to baseline.

Example 4

TNF-α inhibitors

Fc-TNF-α inhibitors. A DNA sequence encoding an in-frame human IgG1 Fc region fused to a TNF-α inhibitory peptide monomer was constructed using standard PCR technology. The Fc and part of the 5-glycine linker molecule were generated by PCR with DNA from 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 inhibitor peptide, provided by PCR primer 2295-89, are shown below:

<td> 1216-52</td><td>AAC</td><td>ATA</td><td>AGT</td><td>ACC</td><td>TGT</td><td>AGG</td><td>ATC</td><td>G.</td><td></td><td></td><td></td><td></td>

<td> 2295-89</td><td>CCG</td><td>CGG</td><td>ATC</td><td>CAT</td><td>TAC</td><td>GGA</td><td>CGG</td><td>TGA</td><td>CCC</td><td>AGA</td><td>GAG</td><td>GTG TTT TTG TAG</td>

<td></td><td>TGC</td><td>GGC</td><td>AGG</td><td>AAG</td><td>TCA</td><td>CCA</td><td>CCA</td><td>CCT</td><td>CCA</td><td>CCT</td><td>TTA</td><td>CCC</td>

Oligonucleotides 2295-89 include the glycine linker and the Fc portion of the template by 22 nucleotides where PCR fuses 2 genes fused together in the correct reading frame.

The PCR gene product (full-length fusion gene) was digested with NdeI and BamHI restriction endonuclease, then ligated into pAMG21 vector and transformed into competent cells of E. coli 2596 strain as described herein for EMP-Fc. Clones were screened for the ability to produce a recombinant protein product and for having a gene fusion with the correct nucleotide sequence. This single clone was selected and designated Amgene strain # 4544.

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The nucleotide and amino acid sequences (SEQ ID NOS: 1055 and 1056) of the fusion protein are shown in Figures 19A and 19B.

TNF-alpha-Fc inhibitor. A DNA sequence encoding a TNF-α inhibitor 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 5 glycine Fc linker. Nucleotides encoding the TNF-α inhibitor peptide were delivered by PCR sense 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:

<td rowspan="2"> 2295-88</td><td rowspan="2">GAA CAC</td><td colspan="4">TAA CAT ATG GAC</td><td rowspan="2">TTC GGC</td><td rowspan="2">CTG GGT</td><td rowspan="2">CCG GGG</td><td rowspan="2">CAC GAC</td><td rowspan="2">TAC AAA</td><td rowspan="2">AAA AAC ACC TCT CTG GGT ACT</td>

<td>CGT</td><td>CCG</td><td>GGT</td><td>GGA</td>

<td> 1200-54</td><td>GTT</td><td>ATT</td><td>GCT</td><td>CAG</td><td>CGG</td><td>TGG</td><td>CA</td><td></td><td></td><td></td><td></td>

Oligonucleotides 2295-88 include a glycine linker and the Fc portion of the template by 24 nucleotides where PCR fuses 2 genes fused together in the correct reading frame.

The PCR gene product (full-length fusion gene) was digested with NdeI and BamHI restriction endonuclease endonuclease, then ligated into the pAMG21 vector and transformed into competent cells of E. coli 2596 strain as described herein for EMP-Fc. Clones were screened for the ability to produce a recombinant protein product and for having a gene fusion with the correct nucleotide sequence. This single clone was selected and designated Amgene strain # 4543.

The nucleotide and amino acid sequences (SEQ ID NOS: 1057 and 1058) of the fusion protein are shown in Figures 20A and 20B.

Expression. Cultures of each pAMG21-Fc-fusion construct in E. coli GM221 were grown at 37 ° C in Luria Broth medium containing 50 mg / ml kanamycin. The induction of gene product expression from the luxPR promoter was achieved after the addition of the synthetic autoinducer lactone N- (3-oxohexanoyl) -DL-homoserine to the culture medium 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 under a microscope for the presence of inclusion bodies and then harvested by centrifugation. Refractillary inclusion bodies were observed in the induced cultures showing that Fc fusions were most likely produced in the insoluble fraction in E. coli. Cell plates were immediately lysed by resuspension in Laemmli sample buffer containing 10% β-mercaptoethanol and analyzed by SDS-PAGE. In each case, an intensely stained Coomassie stained band of the appropriate molecular weight was observed on an SDS-PAGE gel.

Purification of Fc-peptide fusion proteins. The cells were disrupted in water (1/10) by the elevated pressure homogenization method (2 runs at 14,000 PSI = approx. 1.0 x 10⁸ Pa) and the inclusion bodies were collected by centrifugation (4,200 rpm in a J-6B for 1 hour). Inclusion bodies were solubilized in 5 M guanidine, 50 mM Tris, 8 mM DTT, pH 8.7 for 1 hour in a 1/10 ratio. The solubilized mixture was diluted 20-fold in 2 M urea, 50 mM Tris, 160 mM arginine, 3 mM cysteine, pH 8.5. The mixture was stirred overnight under cool conditions and concentrated approximately 10 times by ultrafiltration. It was then diluted 3-fold with 10 mM Tris, 1.5 M urea, pH 9. The pH of this mixture was then adjusted to pH 5 with acetic acid. The pellet was removed by centrifugation and the supernatant was placed on a SP-Sepharose Fast Flow column equilibrated in 20 mM NaAc, 100 mM NaCl, pH 5 (10 mg / ml protein loading, room temperature). The protein was eluted using a 20-column volume of the same buffer with a NaCl concentration gradient from 100 mM NaCl to 500 mM. The column stock was diluted 3-fold and placed on a SP-Sepharose HP column in 20 mM NaAc, 150 mM NaCl, pH 5 (10 mg / ml protein loading, room temperature). The protein was eluted using a 20 column volume of the same buffer with a NaCl concentration gradient from 150 mM NaCl to 400 mM. The peak harvest was collected and filtered.

Characterization of the activity of Fc- TNF-α inhibitor and TNF-α-Fc inhibitor. The binding of these peptide fusion proteins to TNF-α can be characterized by BlAcore by methods available to one of skill in the art, having knowledge of the present patent specification.

Example 5

IL-1 antagonists

Fc- IL-1 antagonist. A DNA sequence encoding an in-frame human IgG1 Fc region fused to an IL-1 antagonist peptide monomer was constructed using standard PCR technology. The Fc portion and the 5-glycine linker portion of the molecule were generated by PCR

From Fc-EMP fusion DNA of 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, provided by PCR primer 2269-70, are shown below:

<td> 1216-52</td><td>AAC</td><td>ATA</td><td>AGT</td><td>ACC</td><td>TGT</td><td>AGG</td><td>ATC</td><td>G.</td><td></td><td></td>

<td> 2269-70</td><td>CCG CAT</td><td>CGG TCG</td><td>ATC AAA</td><td>CAT CCA</td><td>TAC CCA</td><td>AGC CCT</td><td>GGC CCA</td><td>AGA CCT</td><td>GCG TTA</td><td>TAC GGC TGC CAG TAA CCC GGG GTC CCC</td>

Oligonucleotides 2269-70 include the glycine linker and the Fc portion of the template by 22 nucleotides where PCR fuses 2 genes fused together in the correct reading frame.

The PCR gene product (full-length fusion gene) was digested with NdeI and BamHI restriction endonuclease, then ligated into pAMG21 vector and transformed into competent cells of E. coli 2596 strain as described herein for EMP-Fc. Clones were screened for the ability to produce a recombinant protein product and on a gene fusion with the correct nucleotide sequence. This single clone was selected and designated Amgene strain # 4506.

The nucleotide and amino acid sequences (SEQ ID NOS: 1059 and 1060) of the fusion protein are shown in Figures 21A and 21B.

IL-1-Fc antagonist. A DNA sequence encoding 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 5 glycine Fc linker. The nucleotides encoding the IL-1 antagonist peptide were provided by 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:

<td> 2269-69</td><td>GAA</td><td>TAA</td><td>CAT</td><td>ATG</td><td>TTC</td><td>GAA</td><td>TGG</td><td>ACC</td><td>CCG</td><td>GGT</td><td>TAC TGG CAG CCG TAC GCT</td>

<td></td><td>CTG</td><td>CCG</td><td>CTG</td><td>GGT</td><td>GGA</td><td>GGC</td><td>GGT</td><td>GGG</td><td>GAC</td><td>AAA</td><td>ACT</td>

<td> 1200-54</td><td>GTT</td><td>ATT</td><td>GCT</td><td>CAG</td><td>CGG</td><td>TGG</td><td>CA</td><td></td><td></td><td></td><td></td>

Oligonucleotides 2269-69 include the glycine linker and the Fc portion of the template by 24 nucleotides where PCR fuses 2 genes fused together in the correct reading frame.

The PCR gene product (full length fusion gene) was digested with NdeI and BamHI restriction endonuclease, then ligated into the pAMG21 vector and transformed into competent cells of the E. coli 2596 strain as described herein for EMP-Fc. Clones were screened for the ability to produce a recombinant protein product and for the presence of a gene fusion with the correct nucleotide sequence. This single clone was selected and designated as strain Amgen # 4505.

The nucleotide and amino acid sequences (SEQ ID NOS: 1061 and 1062) of the fusion protein are shown in Figures 22A and 22B. Expression and purification were as in the previous examples.

Characterization of IL-1 antagonist Fc-peptide activity and antagonist peptide activity

IL-1-Fc. Competition in binding to the IL-1 receptor between the IL-13, IL-1RA and IL-1 conjugated Fc peptide sequences was performed using the IGEN system. Reaction mixes consisting of 0.4 nM biotin-IL-1R fusion + 15 nM IL-1-TAG + 3 gM competitor + 20 gg / ml conjugated streptavidin beads where IL-1RA, Fc-antagonist of IL-1, antagonist of IL -1-Fc. Competition was tested over a competitor concentration range of 3 gM to 1.5 pM. The results are shown below in Table C.

Table C - Results of competition trials in IL-1 receptor binding

<td></td><td>IL-1pep-Fc</td><td>Fc-IL-1pep</td><td>IL-1ra</td>

<td>KI</td><td> 281,5</td><td> 59,58</td><td> 1,405</td>

<td>EC50</td><td> 530,0</td><td> 112,2</td><td> 2,545</td>

<td>95% confidence interval</td><td></td><td></td><td></td>

<td>EC50</td><td>280.2 to 1002</td><td>54.75 to 229.8</td><td>1.149 to 6.086</td>

<td>KI</td><td>148.9 to 532.5</td><td>29.08 to 122.1</td><td>0.6106 to 3.233</td>

The quality of the match _R2

0,9790

0,9687

0,9602

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Example 6

VEGF antagonists

Fc-VEGF antagonist. A DNA sequence encoding a human IgG1 Fc region fused to a VEGF-mimetic peptide monomer was constructed using standard PCR technology. The pFc-A3 vector and the synthetic VEGF-mimetic peptide gene were the templates for the PCR reactions. A synthetic gene was constructed by the annealing of the following two (2) oligonucleotides (SEQ ID NOS: 1120 and 1121, respectively):

<td> 2293-11</td><td>GTT TGT</td><td>GAA TTT</td><td>CCG GAA</td><td>AAC CGT</td><td>TGT CTG</td><td>GAC</td><td>ATC</td><td>CAT</td><td>GTT</td><td>ATG</td><td>TGG</td><td>GAA</td><td>TGG</td><td>GAA</td>

<td> 2293-12</td><td>CAG</td><td>ACG</td><td>TTC</td><td>AAA</td><td>ACA</td><td>TTC</td><td>CCA</td><td>TTC</td><td>CCA</td><td>CAT</td><td>AAC</td><td>ATG</td><td>GAT</td><td>GTC</td>

<td></td><td>ACA</td><td>GTT</td><td>CGG</td><td>TTC</td><td>AAC</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

The two oligonucleotides were "relaxed" to form the following duplex encoding the amino acid sequence shown below (SEQ ID NOS 1122):

GTTGAACCGAACTGTGACATCCATGTTATGTGGGAATGGGAATGTTTTGAACGTCTG 1 --------- H ----------- h ---------₊---------₊---------₊------- 57

CAACTTGGCTTGACACTGTAGGTACAATACACCCTTACCCTTACAAAACTTGCAGAC a VEPNCDIHVMWEWECPERL This duplex was PCR amplified using 2293-05 and 2293-06 as sense and antisense primers (SEQ ID NOS. 1125 and 1126).

The Fc portion of the molecule was generated by PCR with the pFc-A3 plasmid using primers 2293-03 and 2293-04 as sense and antisense primers (SEQ ID NOS: 1123 and 1124, respectively). A full-length fusion gene was obtained from a third PCR reaction using the outer primers 2293-03 and 2293-06. These primers are shown below:

<td> 2293-03</td><td>ATT ACA</td><td>TGA TGT</td><td>TTC</td><td>TAG</td><td>AAG</td><td>GAG</td><td>GAA</td><td>TAA</td><td>CAT</td><td>ATG</td><td>GAC</td><td>AAA</td><td>ACT</td><td>CAC</td>

<td> 2293-04</td><td>GTC AGA</td><td>ACA CAG</td><td>GTT GGA</td><td>CGG</td><td>TTC</td><td>AAC</td><td>ACC</td><td>ACC</td><td>ACC</td><td>ACC</td><td>ACC</td><td>TTT</td><td>ACC</td><td>CGG</td>

<td> 2293-05</td><td>TCC AAC</td><td>CTG TGT</td><td>TCT GAC</td><td>CCG ATC</td><td>GGT</td><td>AAA</td><td>GGT</td><td>GGT</td><td>GGT</td><td>GGT</td><td>GGT</td><td>GTT</td><td>GAA</td><td>CCG</td>

<td> 2293-06</td><td>CCG</td><td>CGG</td><td>ATC</td><td>CTC</td><td>GAG</td><td>TTA</td><td>CAG</td><td>ACG</td><td>TTC</td><td>AAA</td><td>ACA</td><td>TTC</td><td>CCA</td><td></td>

The PCR gene product (full-length fusion gene) was digested with the restriction endonuclease XbaI and BamHI, then ligated into the pAMG21 vector, and transformed into competent E. coli 2596 host cells as described herein for EMP-Fc. Clones were screened for the ability to produce a recombinant protein product and for the presence of a gene fusion with the correct nucleotide sequence. This single clone was selected and designated Amgene strain # 4523.

The nucleotide and amino acid sequences (SEQ ID NOS: 1063 and 1064) of the fusion protein are shown in Figures 23A and 23B.

VEGF-Fc antagonist. A DNA sequence encoding an in-frame fused VEGF mimetic peptide to the Fc region of human IgG1 was constructed using standard PCR technology. The templates for the PCR reaction were plasmid pFc-A3 and the synthetic gene for the VEGF mimetic peptide described above. The synthetic duplex was amplified by PCR using 2293-07 and 2293-08 as sense and antisense primers (SEQ ID NOS. 1127 and 1128, respectively).

The Fc portion of the molecule was generated by PCR with the pFc-A3 plasmid using primers 2293-09 and 2293-10 as sense and antisense primers (SEQ ID NOS: 1129 and 1130, respectively). A full-length fusion gene was obtained from the third PCR reaction using the external primers 2293-07 and 2293-10. These primers are shown below:

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<td> 2293-07</td><td>ATT TGT</td><td>TGA GAC</td><td>TTC</td><td>TAG</td><td>AAG</td><td>GAG</td><td>GAA</td><td>TAA</td><td>CAT</td><td>ATG</td><td>GTT</td><td>GAA</td><td>CCG</td><td>AAC</td>

<td> 2293-08</td><td>ACA AAA</td><td>TGT ACA</td><td>GTG TTC</td><td>AGT</td><td>TTT</td><td>GTC</td><td>ACC</td><td>ACC</td><td>ACC</td><td>ACC</td><td>ACC</td><td>CAG</td><td>ACG</td><td>TTC</td>

<td> 2293-09</td><td>GAA CAC</td><td>TGT ACA</td><td>TTT TGT</td><td>GAA</td><td>CGT</td><td>CTG</td><td>GGT</td><td>GGT</td><td>GGT</td><td>GGT</td><td>GGT</td><td>GAC</td><td>AAA</td><td>ACT</td>

<td> 2293-10</td><td>CCG</td><td>CGG</td><td>ATC</td><td>CTC</td><td>GAG</td><td>TTA</td><td>TTT</td><td>ACC</td><td>CGG</td><td>AGA</td><td>CAG</td><td>GGA</td><td>GAG</td><td></td>

The PCR gene product (full-length fusion gene) was digested with NdeI and BamHI restriction endonuclease endonuclease, then ligated into the pAMG21 vector and transformed into competent E. coli 2596 host cells as described herein for EMP-Fc. Clones were screened for the ability to produce a recombinant protein product and for the presence of a gene fusion with the correct nucleotide sequence. This single clone was selected and designated Amgene strain # 4524.

The nucleotide and amino acid sequences (SEQ ID NOS: 1065 and 1066) of the fusion protein are shown in Figures 24A and 24B. Expression and purification were as in the previous examples.

Example 7

MMP inhibitors

Fc- MMP inhibitor. A DNA sequence encoding an in-frame human IgG1 Fc region fused to the peptide monomer of the MMP inhibitor was constructed using standard PCR technology. The portion of the molecule comprising the Fc and the 5-glycine linker was generated by PCR with DNA from Fc-TNF-α 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). Nucleotides encoding the MMP inhibitor peptide were provided by PCR primer 2308-67, shown below:

<td> 1216-52</td><td>AAC</td><td>ATA</td><td>AGT</td><td>ACC</td><td>TGT</td><td>AGG</td><td>ATC</td><td>G.</td>

<td> 2308-67</td><td>CCG</td><td>CGG</td><td>ATC</td><td>CAT</td><td>TAG</td><td>CAC</td><td>AGG</td><td>GTG AAA CCC CAG TGG GTG GTG</td>

<td></td><td>CAA</td><td>CCA</td><td>CCA</td><td>CCT</td><td>CCA</td><td>CCT</td><td>TTA</td><td>CCC</td>

Oligonucleotides 2308-57 include the glycine linker and the Fc portion of the template by 22 nucleotides where PCR fuses 2 genes fused together in the correct reading frame.

The PCR gene product (full-length fusion gene) was digested with NdeI and BamHI restriction endonuclease endonuclease, then ligated into the pAMG21 vector and transformed into competent E. coli 2596 host cells as described herein for EMP-Fc. Clones were screened for the ability to produce a recombinant protein product and for the presence of a gene fusion with the correct nucleotide sequence. This single clone was selected and designated Amgene strain # 4597.

The nucleotide and amino acid sequences (SEQ ID NOS: 1067 and 1068) of the fusion protein are shown in Figures 25A and 25B. Expression and purification were as in the previous examples.

MMP-Fc inhibitor. A DNA sequence encoding an MMP inhibitor peptide fused in frame to the Fc region of human IgG1 was constructed using standard PCR technology. The portion of the molecule comprising the Fc and the 5-glycine linker was generated by PCR with DNA from Fc-TNF-α fusion strain # 4543 (see example 4). The nucleotides encoding the MMP inhibitor peptide were delivered by PCR sense 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:

<td rowspan="2"> 2308-66</td><td rowspan="2">GAA GGT</td><td rowspan="2">TAA GGA</td><td colspan="4">CAT ATG TGC ACC</td><td rowspan="2">ACC CAC TGG GGT TTC ACC CTG TGC AAA</td>

<td>GGC</td><td>GGT</td><td>GGG</td><td>GAC</td>

<td> 1200-54</td><td>GTT</td><td>ATT</td><td>GCT</td><td>CAG</td><td>CGG</td><td>TGG</td><td>CA</td>

Oligonucleotides 2269-69 include the glycine linker and the Fc portion of the template by 24 nucleotides where PCR fuses 2 genes fused together in the correct reading frame.

The PCR gene product (full-length fusion gene) was digested with NdeI and BamHI restriction endonuclease, then ligated into the pAMG21 vector and transformed into competent E. coli 2595 host cells as described herein for EMP-Fc. Clones were screened for

The ability to produce a recombinant protein product and to have the gene fusion with the correct nucleotide sequence. This single clone was selected and designated Amgene strain # 4598.

The nucleotide and amino acid sequences (SEQ ID NOS: 1069 and 1070) of the fusion protein are shown in Figures 26A and 25B.

***

After having exhaustively described the invention, it will be understood by those of ordinary skill in the art that many changes and modifications may be made without departing from the spirit and scope of the present invention.

Shortcuts

The abbreviations used throughout the specification have the meanings given below, as long as they are not defined differently in specific circumstances

<td>Ac</td><td>acetyl (used to denote acetylated residues)</td>

<td>AcBpa</td><td>acetylated p-benzoyl-L-phenylalanine</td>

<td>ADCC</td><td>antibody dependent cellular cytotoxicity</td>

<td>Aib</td><td>aminoisobutyric acid</td>

<td>bA</td><td>β-alanine</td>

<td>Bpa</td><td>p-benzoyl-L-phenylalanine</td>

<td>Take</td><td>bromoacetyl [BrCH2C (O)]</td>

<td>BSA</td><td>bovine serum albumin</td>

<td>Bzl</td><td>Benzyl</td>

<td>Cap</td><td>caproic acid</td>

<td>CTL</td><td>Cytotoxic T cells</td>

<td>CTLA4</td><td>4 cytotoxic T cell antigen</td>

<td>DARC</td><td>blood group antigen receptor (Duffy)</td>

<td>DCC</td><td>Dicylcohexylcarbodiimide</td>

<td>Dde</td><td>1- (4,4-dimethyl-2,6-dioxo-cyclohexylidene) ethyl</td>

<td>EMP</td><td>Mimetic Peptide-Erythropoietin</td>

<td>ESI-MS</td><td>Electron spray ionization mass spectrometry</td>

<td>EPO</td><td>Erythropoietin</td>

<td>Fmoc</td><td>fluorenylmethoxycarbonyl</td>

<td>G-CSF</td><td>Granulocyte colony stimulating factor</td>

<td>GH</td><td>Growth hormone</td>

<td>HCT</td><td>hematocrit</td>

<td>HGB</td><td>hemoglobin</td>

<td>hGH</td><td>Human Growth Hormone</td>

<td>HOBt</td><td>1-Hydroxybenzotriazole</td>

<td>HPLC</td><td>high performance liquid chromatography</td>

<td>LOAM</td><td>interleukin</td>

<td>IL-R</td><td>interleukin receptor</td>

<td>IL-1R</td><td>Interleukin-1 receptor</td>

<td>IL-1ra</td><td>interleukin-1 receptor antagonist</td>

<td>Lau</td><td>Laurel acid</td>

<td>LPS</td><td>lipopolysaccharide</td>

<td>LYMPH</td><td>lymphocytes</td>

<td>MALDI-MS</td><td>Matrix-assisted laser desorption ionization mass spectrometry</td>

<td>Me</td><td>methyl</td>

<td>MeO</td><td>methoxy</td>

<td>MHC</td><td>the main histological compatibility complex</td>

<td>MMP</td><td>matrix metalloproteinase</td>

<td>MMPI</td><td>matrix metalloproteinase inhibitor</td>

<td>1-Nap</td><td>1-naptylalanine</td>

<td>NEUT</td><td>neutrophils</td>

<td>NGF</td><td>nerve growth factor</td>

<td>Nle</td><td>norleucine</td>

PL 211 164 B1

<td>NMP</td><td>N-methyl-2-pyrrolidinone</td>

<td>PAGE</td><td>polyacrylamide gel electrophoresis</td>

<td>PBS</td><td>saline with phosphate buffer</td>

<td>Pbf</td><td>2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl</td>

<td>PCR</td><td>polymerase chain reaction</td>

<td>Pec</td><td>pipecolinic acid</td>

<td>PEG</td><td>polyethylene glycol</td>

<td>pGlu</td><td>pyroglutamic acid</td>

<td>Drink</td><td>picolinic acid</td>

<td>PLT</td><td>shallow</td>

<td>pY</td><td>phosphotyrosine</td>

<td>RBC</td><td>Red bloodcells</td>

<td>RBS</td><td>ribosome binding site</td>

<td>RT</td><td>room temperature (25 ° C)</td>

<td>Sar</td><td>sarcosine</td>

<td>SDS</td><td>sodium dodecyl sulfate</td>

<td>STK</td><td>serine-threonine kinase</td>

<td>t-Boc</td><td>tert-butoxycarbonyl</td>

<td>tBu</td><td>tert-butyl</td>

<td>TGF</td><td>tissue growth factor</td>

<td>THF</td><td>humoral factor of the thymus gland</td>

<td>TK</td><td>tyrosine kinase</td>

<td>TMP</td><td>thrombopoietin mimetic peptide</td>

<td>TNF</td><td>tissue necrosis factor</td>

<td>TPO</td><td>Thrombopoietin</td>

<td>TRAIL</td><td>ligand inducing TNF-associated apoptosis</td>

<td>Trt</td><td>trityl</td>

<td>UK</td><td>urokinase</td>

<td>UKR</td><td>urokinase receptor</td>

<td>VEGF</td><td>vascular growth factor of endothelial cells</td>

<td>VIP</td><td>intestinal vasoactive peptide</td>

<td>WBC</td><td>White blood cells</td>

PL 211 164 B1

SEQUENCE LIST &lt;110&gt; AMGEN INC.

&lt;12 0&gt; MODIFIED PEPTIDES AS THERAPEUTICS &lt;130&gt; A-527 &lt;140&gt; PCT / US 99/25044 &lt;141&gt; 1999-10-25 &lt;150&gt; US 09 / 428,082 &lt;151&gt; 1999-10-22 &lt;150&gt; US 60 / 105,371 &lt;151&gt; 1998-10-23 &lt;160&gt; 1143 &lt;170&gt; Patentln version 3.3 &lt;210&gt; 1 &lt;211&gt; 684 &lt;212&gt; DNA &lt;213&gt; HUMAN &lt;220&gt;

&lt;221&gt; CDS &lt;222&gt; (1). . (684) &lt;400&gt; 1

<td>atg Underworld 1</td><td>jacket Asp</td><td>aaa Lys</td><td>act Thr</td><td>cac His 5</td><td>aca Thr</td><td>tgt Cys</td><td>cca Pro</td><td>cct Pro</td><td>tgt Cys 10</td><td>cca Pro</td><td>gct Ala</td><td>ccg Pro</td><td>gaa Glu</td><td>ctc Leu 15</td><td>ctg Leu</td><td> 48</td>

<td>hhg</td><td>gga</td><td>ccg</td><td>tca</td><td>gtc</td><td>ttc</td><td>ctc</td><td>ttc</td><td>ccc</td><td>cca</td><td>aaa</td><td>ccc</td><td>aag</td><td>jacket</td><td>acc</td><td>ctc</td><td> 96</td>

<td>Gly</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>Val</td><td>Phe</td><td>Leu</td><td>Phe</td><td>Pro</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Lys</td><td>Asp</td><td>Thr</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td>

<td>atg</td><td>atc</td><td>tcc</td><td>cgg</td><td>acc</td><td>cct</td><td>gag</td><td>gtc</td><td>aca</td><td>tgc</td><td>gtg</td><td>gtg</td><td>gtg</td><td>jacket</td><td>gtg</td><td>agc</td><td> 144</td>

<td>Underworld</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Val</td><td>Thr</td><td>Cys</td><td>Val</td><td>Val</td><td>Val</td><td>Asp</td><td>Val</td><td>Cheese</td><td></td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td>

<td>cac</td><td>gaa</td><td>jacket</td><td>cct</td><td>gag</td><td>gtc</td><td>aag</td><td>ttc</td><td>aac</td><td>tgg</td><td>trays</td><td>gtg</td><td>jacket</td><td>ggc</td><td>gtg</td><td>gag</td><td> 192</td>

<td>His</td><td>Glu</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Val</td><td>Lys</td><td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Gly</td><td>Val</td><td>Glu</td><td></td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td></td>

<td>gtg</td><td>cat</td><td>aat</td><td>gcc</td><td>aag</td><td>aca</td><td>aag</td><td>ccg</td><td>cgg</td><td>gag</td><td>gag</td><td>cag</td><td>trays</td><td>aac</td><td>agc</td><td>acg</td><td> 240</td>

<td>Val</td><td>His</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Thr</td><td>Lys</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Main</td><td>Tyr</td><td>Asn</td><td>Cheese</td><td>Thr</td><td></td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td>

<td>trays</td><td>cgt</td><td>gtg</td><td>gtc</td><td>agc</td><td>gtc</td><td>ctc</td><td>acc</td><td>gtc</td><td>ctg</td><td>cac</td><td>cag</td><td>jacket</td><td>tgg</td><td>ctg</td><td>aat</td><td> 288</td>

<td>Tyr</td><td>Arg</td><td>Val</td><td>Val</td><td>Cheese</td><td>Val</td><td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>His</td><td>Main</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Asn</td><td></td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td>

<td>ggc</td><td>aag</td><td>gag</td><td>trays</td><td>aag</td><td>tgc</td><td>aag</td><td>gtc</td><td>tcc</td><td>aac</td><td>aaa</td><td>gcc</td><td>ctc</td><td>cca</td><td>gcc</td><td>ccc</td><td> 336</td>

<td>Gly</td><td>Lys</td><td>Glu</td><td>Tyr</td><td>Lys</td><td>Cys</td><td>Lys</td><td>Val</td><td>Cheese</td><td>Asn</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Pro</td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td>

<td>atc</td><td>gag</td><td>aaa</td><td>acc</td><td>atc</td><td>tcc</td><td>aaa</td><td>gcc</td><td>aaa</td><td>hhg</td><td>cag</td><td>ccc</td><td>ega</td><td>gaa</td><td>cca</td><td>cag</td><td> 384</td>

<td>How much</td><td>Glu</td><td>Lys</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Lys</td><td>Ala</td><td>Lys</td><td>Gly</td><td>Main</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Pro</td><td>Main</td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td>

PL 211 164 B1

<td>gtg Val</td><td>trays Tyr 130</td><td>acc Thr</td><td>ctg Leu</td><td>ccc Pro</td><td>cca Pro</td><td>tcc Cheese 135</td><td>cgg Arg</td><td>gen Asp</td><td>gag Glu</td><td>ctg Leu</td><td>acc Thr 140</td><td>aag Lys</td><td>aac Asn</td><td>cag Gin</td><td>gtc. Val</td><td> 432</td>

<td>agc</td><td>ctg</td><td>acc</td><td>tgc</td><td>ctg</td><td>gtc</td><td>aaa</td><td>ggc</td><td>ttc</td><td>tat</td><td>ccc</td><td>agc</td><td>jacket</td><td>atc</td><td>gcc</td><td>gtg</td><td> 480</td>

<td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>How much</td><td>Ala</td><td>Val</td><td></td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td>

<td>gag</td><td>tgg</td><td>gag</td><td>agc</td><td>aat</td><td>hhg</td><td>cag</td><td>ccg</td><td>gag</td><td>aac</td><td>aac</td><td>trays</td><td>aag</td><td>acc</td><td>acg</td><td>cct</td><td> 528</td>

<td>Glu</td><td>Trp</td><td>Glu</td><td>Cheese</td><td>Asn</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td></td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td>

<td>ccc</td><td>gtg</td><td>ctg</td><td>jacket</td><td>tcc</td><td>jacket</td><td>ggc</td><td>tcc</td><td>ttc</td><td>ttc</td><td>ctc</td><td>trays</td><td>agc</td><td>aag</td><td>ctc</td><td>acc</td><td> 576</td>

<td>Pro</td><td>Val</td><td>Leu</td><td>Asp</td><td>Cheese</td><td>Asp</td><td>Gly</td><td>Cheese</td><td>Phe</td><td>Phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Lys</td><td>Leu</td><td>Thr</td><td></td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td>

<td>gtg</td><td>jacket</td><td>aag</td><td>agc</td><td>agg</td><td>tgg</td><td>cag</td><td>cag</td><td>hhg</td><td>aac</td><td>gtc</td><td>ttc</td><td>tca</td><td>tgc</td><td>tcc</td><td>gtg</td><td> 624</td>

<td>Val</td><td>Asp</td><td>Lys</td><td>Cheese</td><td>Arg</td><td>Trp</td><td>Gin</td><td>Gin</td><td>Gly</td><td>Asn</td><td>Val</td><td>Phe</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>Val</td><td></td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td>

<td>atg</td><td>cat</td><td>gag</td><td>gct</td><td>ctg</td><td>cac</td><td>aac</td><td>cac</td><td>trays</td><td>acg</td><td>cag</td><td>aag</td><td>agc</td><td>ctc</td><td>tcc</td><td>ctg</td><td> 672</td>

<td>Underworld</td><td>His</td><td>Glu</td><td>Ala</td><td>Leu</td><td>His</td><td>Asn</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td></td>

<td>tct</td><td>ccg</td><td>ggt</td><td>aaa</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 684</td>

<td>Cheese</td><td>Pro</td><td>Gly</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

225 &lt;210&gt; 2 &lt;211&gt; 228 &lt;212&gt; PRT

<td colspan="16">&lt;213&gt; human sequence</td>

<td colspan="2"> &lt;400&gt; 2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Underworld</td><td>Asp</td><td>Lys</td><td>Thr</td><td>His</td><td>Thr</td><td>Cys</td><td>Pro</td><td>Pro</td><td>Cys</td><td>Pro</td><td>Ala</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Leu</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Gly</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>Val</td><td>Phe</td><td>Leu</td><td>Phe</td><td>Pro</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Lys</td><td>Asp</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Underworld</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Val</td><td>Thr</td><td>Cys</td><td>Val</td><td>Val</td><td>Val</td><td>Asp</td><td>Val</td><td>Cheese</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>His</td><td>Glu</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Val</td><td>Lys</td><td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Gly</td><td>Val</td><td>Glu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>His</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Thr</td><td>Lys</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Tyr</td><td>Asn</td><td>Cheese</td><td>Thr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Tyr</td><td>Arg</td><td>Val</td><td>Val</td><td>Cheese</td><td>Val</td><td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>His</td><td>Gin</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Asn</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Gly</td><td>Lys</td><td>Glu</td><td>Tyr</td><td>Lys</td><td>Cys</td><td>Lys</td><td>Val</td><td>Cheese</td><td>Asn</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Pro</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>How much</td><td>Glu</td><td>Lys</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Lys</td><td>Ala</td><td>Lys</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Pro</td><td>Gin</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Lys</td><td>Asn</td><td>Gin</td><td>Val</td>

130 135 140

PL 211 164 B1

<td>Leu Thr Cheese</td><td>Cys</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>How much</td><td>Ala</td><td>Val</td>

<td> 145</td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Glu Trp Glu</td><td>Cheese</td><td>Asn</td><td>Gly</td><td>Main</td><td>Pro</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Pro Val Leu</td><td>Asp</td><td>Cheese</td><td>Asp</td><td>Gly</td><td>Cheese</td><td>Phe</td><td>Phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Lys</td><td>Leu</td><td>Thr</td>

<td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Val Asp Lys</td><td>Cheese</td><td>Arg</td><td>Trp</td><td>Main</td><td>Main</td><td>Gly</td><td>Asn</td><td>Val</td><td>Phe</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>Val</td>

<td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Met His Glu</td><td>Ala</td><td>Leu</td><td>His</td><td>Asn</td><td>His</td><td>Tyr</td><td>Thr</td><td>Main</td><td>Lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td>

<td> 210</td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Pro Gly cheese 225</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>&lt;210&gt; 3 &lt;211&gt; 36 &lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;213&gt; artificial</td><td colspan="2">sequence</td><td>and</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="11">&lt;223&gt; CONSTRUCT OF A THROMBOPOETHIN-MIMETIC PEPTIDE</td><td></td><td></td><td></td>

&lt;220&gt;

&lt;221&gt; other characteristics &lt;222&gt; (18). . (18) &lt;223&gt; 5000 Dalton methoxy polyethylene glycol -sulfoacetyl group attached to side chain &lt;400&gt; 3

<td rowspan="3">How much Glu 1 Gly Lys Ala Ala</td><td rowspan="3">Gly Gly Arg 35</td><td rowspan="3">Pro Gly twenty Ala</td><td colspan="3" rowspan="2">Thr Leu Arg Gln Trp 5</td><td colspan="5">Leu Ala Ala Arg Ala</td><td colspan="2" rowspan="2">Gly Gly 15</td>

<td colspan="4"> 10</td><td rowspan="2">Main thirty</td>

<td>Gly Gly How many</td><td>Glu</td><td>Gly 25</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Arg</td><td>Trp</td><td>Leu</td>

<td> &lt;210&gt;</td><td> 4</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;211&gt;</td><td> 36</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;213&gt;</td><td colspan="2">Artificial</td><td>sequence a</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td colspan="6">THE CONSTRUCT OF THE THROMBOPOETHINE PEPTIDE</td><td colspan="3">MIMETIC</td><td></td><td></td><td></td>

&lt;220&gt;

&lt;221&gt; other characteristics &lt;222&gt; (18) .. (18) &lt;223&gt; methoxy polyethylene glycol (5000 Dalton) -succinimidyl group attached to the side chain &lt;400&gt; 4

Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly Gly 15 10 15

PL 211 164 B1

Gly Cys Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu 20 25 30

Ala Ala Arg Ala 35 &lt;210&gt; 5 &lt;211&gt; 794 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Fc-TMP &lt;220&gt;

&lt;221&gt; CDS &lt;222&gt; (39) .. (779) &lt;400&gt; 5 tctagatttg ttttaactaa ttaaaggagg aataacat atg gac aaa act cac aca 56

Met Asp Lys Thr His Thr

<td>tgt</td><td>cca</td><td>cct</td><td>tgt</td><td>cca</td><td>gct</td><td>ccg</td><td>gaa</td><td>ctc</td><td>ctg</td><td>1 hhg</td><td>gga</td><td>ccg</td><td>tca</td><td>5 gtc</td><td>ttc</td><td> 104</td>

<td>Cys</td><td>Pro</td><td>Pro</td><td>Cys</td><td>Pro</td><td>Ala</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>Val</td><td>Phe</td><td></td>

<td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td><td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td>

<td>ctc</td><td>ttc</td><td>ccc</td><td>cca</td><td>aaa</td><td>ccc</td><td>aag</td><td>jacket</td><td>acc</td><td>ctc</td><td>atg</td><td>atc</td><td>tcc</td><td>cgg</td><td>acc</td><td>cct</td><td> 152</td>

<td>Leu</td><td>Phe</td><td>Pro</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Lys</td><td>Asp</td><td>Thr</td><td>Leu</td><td>Underworld</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Pro</td><td></td>

<td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td>

<td>gag</td><td>gtc</td><td>aca</td><td>tgc</td><td>gtg</td><td>gtg</td><td>gtg</td><td>jacket</td><td>gtg</td><td>agc</td><td>cac</td><td>gaa</td><td>jacket</td><td>cct</td><td>gag</td><td>gtc</td><td> 200</td>

<td>Glu</td><td>Val</td><td>Thr</td><td>Cys</td><td>Val</td><td>Val</td><td>Val</td><td>Asp</td><td>Val</td><td>Cheese</td><td>His</td><td>Glu</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Val</td><td></td>

<td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td></td>

<td>aag</td><td>ttc</td><td>aac</td><td>tgg</td><td>trays</td><td>gtg</td><td>jacket</td><td>ggc</td><td>gtg</td><td>gag</td><td>gtg</td><td>cat</td><td>aat</td><td>gcc</td><td>aag</td><td>aca</td><td> 248</td>

<td>Lys</td><td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Gly</td><td>Val</td><td>Glu</td><td>Val</td><td>His</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Thr</td><td></td>

<td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td>

<td>aag</td><td>ccg</td><td>cgg</td><td>gag</td><td>gag</td><td>cag</td><td>trays</td><td>aac</td><td>agc</td><td>acg</td><td>trays</td><td>cgt</td><td>gtg</td><td>gtc</td><td>agc</td><td>gtc</td><td> 296</td>

<td>Lys</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Main</td><td>Tyr</td><td>Asn</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Arg</td><td>Val</td><td>Val</td><td>Cheese</td><td>Val</td><td></td>

<td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td>

<td>ctc</td><td>acc</td><td>gtc</td><td>ctg</td><td>cac</td><td>cag</td><td>jacket</td><td>tgg</td><td>ctg</td><td>aat</td><td>ggc</td><td>aag</td><td>gag</td><td>trays</td><td>aag</td><td>tgc</td><td> 344</td>

<td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>His</td><td>Main</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Asn</td><td>Gly</td><td>Lys</td><td>Glu</td><td>Tyr</td><td>Lys</td><td>Cys</td><td></td>

<td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td>

<td>aag</td><td>gtc</td><td>tcc</td><td>aac</td><td>aaa</td><td>gcc</td><td>ctc</td><td>cca</td><td>gcc</td><td>ccc</td><td>atc</td><td>gag</td><td>aaa</td><td>acc</td><td>atc</td><td>tcc</td><td> 392</td>

<td>Lys</td><td>Val</td><td>Cheese</td><td>Asn</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Pro</td><td>How much</td><td>Glu</td><td>Lys</td><td>Thr</td><td>How much</td><td>Cheese</td><td></td>

<td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td>

<td>aaa</td><td>gcc</td><td>aaa</td><td>hhg</td><td>cag</td><td>ccc</td><td>ega</td><td>gaa</td><td>cca</td><td>cag</td><td>gtg</td><td>trays</td><td>acc</td><td>ctg</td><td>ccc</td><td>cca</td><td> 440</td>

<td>Lys</td><td>Ala</td><td>Lys</td><td>Gly</td><td>Main</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Pro</td><td>Main</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td><td></td>

<td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td></td>

<td>tcc</td><td>cgg</td><td>gen</td><td>gag</td><td>ctg</td><td>acc</td><td>aag</td><td>aac</td><td>cag</td><td>gtc</td><td>agc</td><td>ctg</td><td>acc</td><td>tgc</td><td>ctg</td><td>gtc</td><td> 488</td>

<td>Cheese</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Lys</td><td>Asn</td><td>Main</td><td>Val</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td></td>

<td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td>

<td>aaa</td><td>ggc</td><td>ttc</td><td>tat</td><td>ccc</td><td>agc</td><td>jacket</td><td>atc</td><td>gcc</td><td>gtg</td><td>gag</td><td>tgg</td><td>gag</td><td>agc</td><td>aat</td><td>hhg</td><td> 536</td>

<td>Lys</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>How much</td><td>Ala</td><td>Val</td><td>Glu</td><td>Trp</td><td>Glu</td><td>Cheese</td><td>Asn</td><td>Gly</td><td></td>

<td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td>

PL 211 164 B1

<td>cag Gin</td><td>ccg Pro</td><td>gag aac Glu Asn 170</td><td>aac Asn</td><td>trays Tyr</td><td>aag Lys</td><td>acc Thr</td><td>acg Thr 175</td><td>cct Pro</td><td>ccc Pro</td><td>gtg Val</td><td>ctg gac Leu Asp 180</td><td>tcc Cheese</td><td>gac · Asp</td><td> 584</td>

<td>ggc</td><td>tcc</td><td>ttc ttc</td><td>ctc</td><td>trays</td><td>agc</td><td>aag</td><td>ctc</td><td>acc</td><td>gtg</td><td>jacket</td><td>aag agc</td><td>agg</td><td>tgg</td><td> 632</td>

<td>Gly</td><td>Cheese</td><td>Phe Phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys Ser</td><td>Arg</td><td>Trp</td><td></td>

<td></td><td></td><td> 185</td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td><td></td><td></td><td></td>

<td>cag</td><td>cag</td><td>ggg aac</td><td>gtc</td><td>ttc</td><td>tca</td><td>tgc</td><td>tcc</td><td>gtg</td><td>atg</td><td>cat</td><td>gag gct</td><td>ctg</td><td>cac</td><td> 680</td>

<td>Gin</td><td>Gin</td><td>Gly Asn</td><td>Val</td><td>Phe</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>Val</td><td>Underworld</td><td>His</td><td>Glu Ala</td><td>Leu</td><td>His</td><td></td>

<td></td><td> 200</td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td><td></td><td></td><td></td><td></td>

<td>aac</td><td>cac</td><td>tac acg</td><td>cag</td><td>aag</td><td>agc</td><td>ctc</td><td>tcc</td><td>ctg</td><td>tct</td><td>ccg</td><td>ggt aaa</td><td>ggt</td><td>gga</td><td> 728</td>

<td>Asn</td><td>His</td><td>Tyr Thr</td><td>Gin</td><td>Lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Gly Lys</td><td>Gly</td><td>Gly</td><td></td>

<td> 215</td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td><td></td><td></td><td></td><td> 230 '</td><td></td>

<td>ggt</td><td>ggt</td><td>ggt atc</td><td>gaa</td><td>ggt</td><td>ccg</td><td>act</td><td>ctg</td><td>cgt</td><td>cag</td><td>tgg</td><td>ctg gct</td><td>gct</td><td>cgt</td><td> 776</td>

<td>Gly</td><td>Gly</td><td>Gly How Much</td><td>Glu</td><td>Gly</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Arg</td><td>Gin</td><td>Trp</td><td>Leu Ala</td><td>Ala</td><td>Arg</td><td></td>

<td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td><td></td><td></td><td> 245</td><td></td><td></td>

<td>gct</td><td colspan="2">taatctcgag &lt;</td><td colspan="2">gatcc</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 794</td>

Ala &lt;210&gt; 6 &lt;211&gt; 247 &lt;212&gt; PRT

<td>&lt;213&gt; artificial</td><td>sequence a</td>

<td> &lt;220&gt;</td><td></td>

<td>&lt;223&gt; Construct</td><td>. synthetic</td>

<td> &lt;400&gt; 6</td><td></td>

<td>Met Asp Lys Thr</td><td>His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu</td>

<td> 1</td><td> 5 10 15</td>

<td>Gly Gly Pro Ser</td><td>Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu</td>

<td> 20</td><td> 25 30</td>

<td>Met Ile Cheese Arg</td><td>Thr Pro Glu Val Thr Cys Val Val Val Val Asp Val Ser</td>

<td> 35</td><td> 40 45</td>

<td>His Glu Asp Pro</td><td>Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu</td>

<td> 50</td><td> 55 60</td>

<td>Val His Asn Ala</td><td>Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr</td>

<td> 65</td><td> 70 75 80</td>

<td>Tyr Arg Val Val</td><td>Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn</td>

<td></td><td> 85 90 95</td>

<td>Gly Lys Glu Tyr</td><td>Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro</td>

<td> 100</td><td> 105 110</td>

<td>How many Glu Lys Thr</td><td>Ile Che Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin</td>

<td> 115</td><td> 120 125</td>

<td>Val Tyr Thr Leu</td><td>Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val</td>

<td> 130</td><td> 135 140</td>

<td>Leu Thr Cys</td><td>Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val</td>

145 150 155 160

PL 211 164 B1

<img file="PL211164B1_D0002.tif" />

PL 211 164 B1

<td>aaa Lys</td><td>gcc Ala 120</td><td>aaa Lys</td><td>hhg Gly</td><td>cag Gin</td><td>ccc Pro</td><td>cga Arg 125</td><td>gaa Glu</td><td>cca Pro</td><td>cag Gin</td><td>gtg Val</td><td>trays Tyr 130</td><td>acc Thr</td><td>ctg Leu</td><td>ccc Pro</td><td>cca Pro</td><td> 440</td>

<td>tcc</td><td>cgg</td><td>gen</td><td>gag</td><td>ctg</td><td>acc</td><td>aag</td><td>aac</td><td>cag</td><td>gtc</td><td>agc</td><td>ctg</td><td>acc</td><td>tgc</td><td>ctg</td><td>gtc</td><td> 488</td>

<td>Cheese 135</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Thr 140</td><td>Lys</td><td>Asn</td><td>Gin</td><td>Val</td><td>Cheese 145</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val 150</td><td></td>

<td>aaa</td><td>ggc</td><td>ttc</td><td>tat</td><td>ccc</td><td>agc</td><td>jacket</td><td>atc</td><td>gcc</td><td>gtg</td><td>gag</td><td>tgg</td><td>gag</td><td>agc</td><td>aat</td><td>hhg</td><td> 536</td>

<td>Lys</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro 155</td><td>Cheese</td><td>Asp</td><td>How much</td><td>Ala</td><td>Val 160</td><td>Glu</td><td>Trp</td><td>Glu</td><td>Cheese</td><td>Asn 165</td><td>Gly</td><td></td>

<td>cag</td><td>ccg</td><td>gag</td><td>aac</td><td>aac</td><td>trays</td><td>aag</td><td>acc</td><td>acg</td><td>cct</td><td>ccc</td><td>gtg</td><td>ctg</td><td>jacket</td><td>tcc</td><td>jacket</td><td> 584</td>

<td>Gin</td><td>Pro</td><td>Glu</td><td>Asn 170</td><td>Asn</td><td>Tyr</td><td>Lys</td><td>Thr</td><td>Thr 175</td><td>Pro</td><td>Pro</td><td>Val</td><td>Leu</td><td>Asp 180</td><td>Cheese</td><td>Asp</td><td></td>

<td>ggc</td><td>tcc</td><td>ttc</td><td>ttc</td><td>ctc</td><td>trays</td><td>agc</td><td>aag</td><td>ctc</td><td>acc</td><td>gtg</td><td>jacket</td><td>aag</td><td>agc</td><td>agg</td><td>tgg</td><td> 632</td>

<td>Gly</td><td>Cheese</td><td>Phe 185</td><td>Phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Lys 190</td><td>Leu</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys 195</td><td>Cheese</td><td>Arg</td><td>Trp</td><td></td>

<td>cag</td><td>cag</td><td>hhg</td><td>aac</td><td>gtc</td><td>ttc</td><td>tca</td><td>tgc</td><td>tcc</td><td>gtg</td><td>atg</td><td>cat</td><td>gag</td><td>gct</td><td>ctg</td><td>cac</td><td> 680</td>

<td>Gin</td><td>Gin 200</td><td>Gly</td><td>Asn</td><td>Val</td><td>Phe</td><td>Cheese 205</td><td>Cys</td><td>Cheese</td><td>Val</td><td>Underworld</td><td>His 210</td><td>Glu</td><td>Ala</td><td>Leu</td><td>His</td><td></td>

<td>aac</td><td>cac</td><td>trays</td><td>acg</td><td>cag</td><td>aag</td><td>agc</td><td>ctc</td><td>tcc</td><td>ctg</td><td>tct</td><td>ccg</td><td>ggt</td><td>aaa</td><td>ggt</td><td>gga</td><td> 728</td>

<td>Asn 215</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Lys 220</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese 225</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Gly</td><td>Gly 230</td><td></td>

<td>ggt</td><td>ggt</td><td>ggt</td><td>atc</td><td>gaa</td><td>ggt</td><td>ccg</td><td>act</td><td>ctg</td><td>cgt</td><td>cag</td><td>tgg</td><td>ctg</td><td>gct</td><td>gct</td><td>cgt</td><td> 776</td>

<td>Gly</td><td>Gly</td><td>Gly</td><td>How much</td><td>Glu 235</td><td>Gly</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Arg 240</td><td>Gin</td><td>Trp</td><td>Leu</td><td>Ala</td><td>Ala 245</td><td>Arg</td><td></td>

<td>gct</td><td>ggt</td><td>ggt</td><td>gga</td><td>ggt</td><td>ggc</td><td>ggc</td><td>gga</td><td>ggt</td><td>att</td><td>gag</td><td>ggc</td><td>cca</td><td>acc</td><td>ctt</td><td>cgc</td><td> 824</td>

<td>Ala</td><td>Gly</td><td>Gly</td><td>Gly 250</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly 255</td><td>How much</td><td>Glu</td><td>Gly</td><td>Pro</td><td>Thr 260</td><td>Leu</td><td>Arg</td><td></td>

caa tgg ctt gca gca cgc gcataatctc gaggatccg 861

Gin Trp Leu Ala Ala Arg

265 &lt;210&gt; 8 &lt;211&gt; 268 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; synthetic construct &lt;400&gt; 8

<td>Underworld 1</td><td>Asp</td><td>Lys</td><td>Thr</td><td>His 5</td><td>Thr</td><td>Cys</td><td>Pro</td>

<td>Gly</td><td>Gly</td><td>Pro</td><td>Cheese twenty</td><td>Val</td><td>Phe</td><td>Leu</td><td>Phe</td>

<td>Underworld</td><td>How much</td><td>Cheese 35</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Val 40</td>

<td>His</td><td>Glu 50</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Val</td><td>Lys 55</td><td>Phe</td>

<td>Pro</td><td>Cys 10</td><td>Pro</td><td>Ala</td><td>Pro</td><td>Glu</td><td>Leu 15</td><td>Leu</td>

<td>Pro 25</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Lys</td><td>Asp thirty</td><td>Thr</td><td>Leu</td>

<td>Thr</td><td>Cys</td><td>Val</td><td>Val</td><td>Val 45</td><td>Asp</td><td>Val</td><td>Cheese</td>

<td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Gly</td><td>Val</td><td>Glu</td>

PL 211 164 B1

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr 65 70 75 80

Tire Arg

Gly Lys

Val Val

Val Leu Thr Val Leu His Gin Asp cheese 85 90

Glu Tyr 100

Lys Cys Lys Val

Asn Lys Ala Leu cheese 105

Trp Leu Asn 95

Pro Ala Pro 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gin Pro Arg 115 120 125

Glu Pro Gin

Val Tyr 130

Thr Leu Pro Pro

Ser Arg Asp Glu Leu Thr Lys 135 140

Asn Gin Val

Leu Thr Cys Cheese 145

Glu Trp Glu Ser

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 150 155 160

Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175

Pro Val Leu Asp Ser Asp Gly Cheese Phe Phe Leu Tyr Ser 180 185

Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 195 200 205

Lys Leu Thr 190

Cys Ser Val

Met His 210

Glu Ala Leu His

Asn His Tyr Thr Gin Lys Ser 215 220

Leu Ser Leu

Pro cheese 225

Gly Lys

Gly Gly 230

Gin Trp Leu Ala

Glu Gly Pro Thr 260

Ala Arg 245

Gly Gly

Ala Gly

Leu Arg Gin Trp

Gly Ile Glu Gly Pro 235

Gly Gly Gly Gly Gly 250

Leu Ala Ala Arg 265

Thr Leu Arg 240

Gly Gly Ile 255

<td> &lt;210&gt;</td><td> 9</td>

<td> &lt;211&gt;</td><td> 855</td>

<td> &lt;212&gt;</td><td>GOUT</td>

<td> &lt;213&gt;</td><td>Artificial sequence</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>TMP-TMP-Fc</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;221&gt;</td><td>CDS</td>

<td> &lt;222&gt;</td><td> (39) . . (845)</td>

<td> &lt;400&gt;</td><td> 9</td>

tctagatttg ttttaactaa ttaaaggagg aataacat atg atc gaa ggt ccg act Met Ile Glu Gly Pro Thr 1 5 ctg cgt cag tgg ctg gct gct cgt gct ggc ggt ggt ggc gga ggg ggt Leu Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

15 20

104

PL 211 164 B1

<td>ggc Gly</td><td>att How much</td><td>gag Glu 25</td><td>ggc Gly</td><td>cca Pro</td><td>acc Thr</td><td>ctt Leu</td><td>cgc Arg thirty</td><td>all Gin</td><td>tgg Trp</td><td>ctt Leu</td><td>gca Ala</td><td>gca Ala 35</td><td>cgc Arg</td><td>gca Ala</td><td>hhg Gly</td><td> 152</td>

<td>gga</td><td>ggc</td><td>ggt</td><td>hhg</td><td>jacket</td><td>aaa</td><td>act</td><td>cac</td><td>aca</td><td>tgt</td><td>cca</td><td>cct</td><td>tgc</td><td>cca</td><td>gca</td><td>cct</td><td> 200</td>

<td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Asp</td><td>Lys</td><td>Thr</td><td>His</td><td>Thr</td><td>Cys</td><td>Pro</td><td>Pro</td><td>Cys</td><td>Pro</td><td>Ala</td><td>Pro</td><td></td>

<td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td></td>

<td>gaa</td><td>ctc</td><td>ctg</td><td>hhg</td><td>gga</td><td>ccg</td><td>tca</td><td>gtt</td><td>ttc</td><td>ctc</td><td>ttc</td><td>ccc</td><td>cca</td><td>aaa</td><td>ccc</td><td>aag</td><td> 248</td>

<td>Glu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>Val</td><td>Phe</td><td>Leu</td><td>Phe</td><td>Pro</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Lys</td><td></td>

<td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td>

<td>jacket</td><td>acc</td><td>ctc</td><td>atg</td><td>atc</td><td>tcc</td><td>cgg</td><td>acc</td><td>cct</td><td>gag</td><td>gtc</td><td>aca</td><td>tgc</td><td>gtg</td><td>gtg</td><td>gtg</td><td> 296</td>

<td>Asp</td><td>Thr</td><td>Leu</td><td>Underworld</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Val</td><td>Thr</td><td>Cys</td><td>Val</td><td>Val</td><td>Val</td><td></td>

<td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td>

<td>jacket</td><td>gtg</td><td>agc</td><td>cac</td><td>gaa</td><td>jacket</td><td>cct</td><td>gag</td><td>gtc</td><td>aag</td><td>ttc</td><td>aac</td><td>tgg</td><td>trays</td><td>gtg</td><td>jacket</td><td> 344</td>

<td>Asp</td><td>Val</td><td>Cheese</td><td>His</td><td>Glu</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Val</td><td>Lys</td><td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp</td><td></td>

<td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td>

<td>ggc</td><td>gtg</td><td>gag</td><td>gtg</td><td>cat</td><td>aat</td><td>gcc</td><td>aag</td><td>aca</td><td>aag</td><td>ccg</td><td>cgg</td><td>gag</td><td>gag</td><td>cag</td><td>trays</td><td> 392</td>

<td>Gly</td><td>Val</td><td>Glu</td><td>Val</td><td>His</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Thr</td><td>Lys</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Tyr</td><td></td>

<td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td>

<td>aac</td><td>agc</td><td>acg</td><td>trays</td><td>cgt</td><td>gtg</td><td>gtc</td><td>agc</td><td>gtc</td><td>ctc</td><td>acc</td><td>gtc</td><td>ctg</td><td>cac</td><td>cag</td><td>jacket</td><td> 440</td>

<td>Asn</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Arg</td><td>Val</td><td>Val</td><td>Cheese</td><td>Val</td><td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>His</td><td>Gin</td><td>Asp</td><td></td>

<td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td></td>

<td>tgg</td><td>ctg</td><td>aat</td><td>ggc</td><td>aag</td><td>gag</td><td>trays</td><td>aag</td><td>tgc</td><td>aag</td><td>gtc</td><td>tcc</td><td>aac</td><td>aaa</td><td>gcc</td><td>ctc</td><td> 488</td>

<td>Trp</td><td>Leu</td><td>Asn</td><td>Gly</td><td>Lys</td><td>Glu</td><td>Tyr</td><td>Lys</td><td>Cys</td><td>Lys</td><td>Val</td><td>Cheese</td><td>Asn</td><td>Lys</td><td>Ala</td><td>Leu</td><td></td>

<td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td>

<td>cca</td><td>gcc</td><td>ccc</td><td>atc</td><td>gag</td><td>aaa</td><td>acc</td><td>atc</td><td>tcc</td><td>aaa</td><td>gcc</td><td>aaa</td><td>hhg</td><td>cag</td><td>ccc</td><td>ega</td><td> 536</td>

<td>Pro</td><td>Ala</td><td>Pro</td><td>How much</td><td>Glu</td><td>Lys</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Lys</td><td>Ala</td><td>Lys</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Arg</td><td></td>

<td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td>

<td>gaa</td><td>cca</td><td>cag</td><td>gtg</td><td>trays</td><td>acc</td><td>ctg</td><td>ccc</td><td>cca</td><td>tcc</td><td>cgg</td><td>gen</td><td>gag</td><td>ctg</td><td>acc</td><td>aag</td><td> 584</td>

<td>Glu</td><td>Pro</td><td>Gin</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Lys</td><td></td>

<td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td>

<td>aac</td><td>cag</td><td>gtc</td><td>agc</td><td>ctg</td><td>acc</td><td>tgc</td><td>ctg</td><td>gtc</td><td>aaa</td><td>ggc</td><td>ttc</td><td>tat</td><td>ccc</td><td>agc</td><td>jacket</td><td> 632</td>

<td>Asn</td><td>Gin</td><td>Val</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>Asp</td><td></td>

<td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td>

<td>atc</td><td>gcc</td><td>gtg</td><td>gag</td><td>tgg</td><td>gag</td><td>agc</td><td>aat</td><td>hhg</td><td>cag</td><td>ccg</td><td>gag</td><td>aac</td><td>aac</td><td>trays</td><td>aag</td><td> 680</td>

<td>How much</td><td>Ala</td><td>Val</td><td>Glu</td><td>Trp</td><td>Glu</td><td>Cheese</td><td>Asn</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>Lys</td><td></td>

<td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td><td></td><td></td><td></td><td></td><td></td>

<td>acc</td><td>acg</td><td>cct</td><td>ccc</td><td>gtg</td><td>ctg</td><td>jacket</td><td>tcc</td><td>jacket</td><td>ggc</td><td>tcc</td><td>ttc</td><td>ttc</td><td>ctc</td><td>trays</td><td>agc</td><td> 728</td>

<td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>Val</td><td>Leu</td><td>Asp</td><td>Cheese</td><td>Asp</td><td>Gly</td><td>Cheese</td><td>Phe</td><td>Phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td></td>

<td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td>

<td>aag</td><td>ctc</td><td>acc</td><td>gtg</td><td>jacket</td><td>aag</td><td>agc</td><td>agg</td><td>tgg</td><td>cag</td><td>cag</td><td>hhg</td><td>aac</td><td>gtc</td><td>ttc</td><td>tca</td><td> 776</td>

<td>Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys</td><td>Cheese</td><td>Arg</td><td>Trp</td><td>Gin</td><td>Gin</td><td>Gly</td><td>Asn</td><td>Val</td><td>Phe</td><td>Cheese</td><td></td>

<td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td>

<td>tgc</td><td>tcc</td><td>gtg</td><td>atg</td><td>cat</td><td>gag</td><td>gct</td><td>ctg</td><td>cac</td><td>aac</td><td>cac</td><td>trays</td><td>acg</td><td>cag</td><td>aag</td><td>agc</td><td> 824</td>

<td>Cys</td><td>Cheese</td><td>Val</td><td>Underworld</td><td>His</td><td>Glu</td><td>Ala</td><td>Leu</td><td>His</td><td>Asn</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Lys</td><td>Cheese</td><td></td>

<td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td><td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td>

<td>ctc</td><td>tcc</td><td>ctg</td><td>tct</td><td>ccg</td><td>ggt</td><td>aaa</td><td colspan="3">taatggatcc</td><td></td><td></td><td></td><td></td><td></td><td></td><td> 855</td>

<td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

265

PL 211 164 B1 &lt;210&gt; 10 &lt;211&gt; 269 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; synthetic construct &lt;400&gt; 10

<td rowspan="2">Underworld 1</td><td rowspan="2">How much</td><td colspan="14">Glu Gly Pro Thr Leu Arg Gin Trp Leu Ala Ala Arg Ala Gly</td>

<td colspan="5"> 5</td><td colspan="4"> 10</td><td colspan="5"> 15</td>

<td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>How much</td><td>Glu</td><td>Gly</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Arg</td><td>Gin</td><td>Trp</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>Ala</td><td>Ala</td><td>Arg</td><td>Ala</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Asp</td><td>Lys</td><td>Thr</td><td>His</td><td>Thr</td><td>Cys</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Pro</td><td>Pro</td><td>Cys</td><td>Pro</td><td>Ala</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>Val</td><td>Phe</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Phe</td><td>Pro</td><td>Pro</td><td>Lys</td><td>Pro</td><td>Lys</td><td>Asp</td><td>Thr</td><td>Leu</td><td>Underworld</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Thr</td><td>Pro</td><td>Glu</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Val</td><td>Thr</td><td>Cys</td><td>Val</td><td>Val</td><td>Val</td><td>Asp</td><td>Val</td><td>Cheese</td><td>His</td><td>Glu</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Val</td><td>Lys</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Gly</td><td>Val</td><td>Glu</td><td>Val</td><td>His</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Thr</td><td>Lys</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Pro</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Tyr</td><td>Asn</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Arg</td><td>Val</td><td>Val</td><td>Cheese</td><td>Val</td><td>Leu</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Thr</td><td>Val</td><td>Leu</td><td>His</td><td>Gin</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Asn</td><td>Gly</td><td>Lys</td><td>Glu</td><td>Tyr</td><td>Lys</td><td>Cys</td><td>Lys</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Cheese</td><td>Asn</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Pro</td><td>How much</td><td>Glu</td><td>Lys</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Lys</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Ala</td><td>Lys</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Pro</td><td>Gin</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Arg</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Lys</td><td>Asn</td><td>Gin</td><td>Val</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td>Lys</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>How much</td><td>Ala</td><td>Val</td><td>Glu</td><td>Trp</td><td>Glu</td><td>Cheese</td><td>Asn</td><td>Gly</td><td>Gin</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Pro</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>Val</td><td>Leu</td><td>Asp</td><td>Cheese</td><td>Asp</td><td>Gly</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Phe</td><td>Phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys</td><td>Cheese</td><td>Arg</td><td>Trp</td><td>Gin</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Gin</td><td>Gly</td><td>Asn</td><td>Val</td><td>Phe</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>Val</td><td>Underworld</td><td>His</td><td>Glu</td><td>Ala</td><td>Leu</td><td>His</td><td>Asn</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Lys</td><td></td><td></td><td></td>

260 265 &lt;210&gt; 11 &lt;211&gt; 789 &lt;212&gt; DNA

&lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; TMP-Fc &lt;220&gt;

&lt;221&gt; CDS &lt;222&gt; (39) .. (779) &lt;400&gt; 11 tctagatttg ttttaactaa ttaaaggagg aataacat atg atc gaa ggt ccg act 56

Met Ile Glu Gly Pro Thr 1 5

<td>ctg Leu</td><td>cgt Arg</td><td>cag Gin</td><td>tgg Trp 10</td><td>ctg Leu</td><td>get Ala</td><td>get Ala</td><td>cgt Arg</td><td>get Ala 15</td><td>ggt Gly</td><td>gga Gly</td><td>ggc Gly</td><td>ggt Gly</td><td>hhg Gly twenty</td><td>jacket Asp</td><td>aaa Lys</td><td> 104</td>

<td>act</td><td>cac</td><td>aca</td><td>tgt</td><td>cca</td><td>cct</td><td>tgc</td><td>cca</td><td>gca</td><td>cct</td><td>gaa</td><td>ctc</td><td>ctg</td><td>hhg</td><td>gga</td><td>ccg</td><td> 152</td>

<td>Thr</td><td>His</td><td>Thr 25</td><td>Cys</td><td>Pro</td><td>Pro</td><td>Cys</td><td>Pro thirty</td><td>Ala</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Leu 35</td><td>Gly</td><td>Gly</td><td>Pro</td><td></td>

<td>tea</td><td>gtt</td><td>ttc</td><td>ctc</td><td>ttc</td><td>ccc</td><td>cca</td><td>aaa</td><td>ccc</td><td>aag</td><td>jacket</td><td>acc</td><td>ctc</td><td>atg</td><td>atc</td><td>tcc</td><td> 200</td>

<td>Cheese</td><td>Val 40</td><td>Phe</td><td>Leu</td><td>Phe</td><td>Pro</td><td>Pro 45</td><td>Lys</td><td>Pro</td><td>Lys</td><td>Asp</td><td>Thr 50</td><td>Leu</td><td>Underworld</td><td>How much</td><td>Cheese</td><td></td>

<td>cgg</td><td>acc</td><td>cct</td><td>gag</td><td>gtc</td><td>aca</td><td>tgc</td><td>gtg</td><td>gtg</td><td>gtg</td><td>jacket</td><td>gtg</td><td>age</td><td>cac</td><td>gaa</td><td>jacket</td><td> 248</td>

<td>Arg 55</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Val</td><td>Thr 60</td><td>Cys</td><td>Val</td><td>Val</td><td>Val</td><td>Asp 65</td><td>Val</td><td>Cheese</td><td>His</td><td>Glu</td><td>Asp 70</td><td></td>

<td>cct</td><td>gag</td><td>gtc</td><td>aag</td><td>ttc</td><td>aac</td><td>tgg</td><td>trays</td><td>gtg</td><td>jacket</td><td>ggc</td><td>gtg</td><td>gag</td><td>gtg</td><td>cat</td><td>aat</td><td> 296</td>

<td>Pro</td><td>Glu</td><td>Val</td><td>Lys</td><td>Phe 75</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp 80</td><td>Gly</td><td>Val</td><td>Glu</td><td>Val</td><td>His 85</td><td>Asn</td><td></td>

<td>gee</td><td>aag</td><td>aca</td><td>aag</td><td>ccg</td><td>cgg</td><td>gag</td><td>gag</td><td>cag</td><td>trays</td><td>aac</td><td>age</td><td>acg</td><td>trays</td><td>cgt</td><td>gtg</td><td> 344</td>

<td>Ala</td><td>Lys</td><td>Thr</td><td>Lys 90</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Gin 95</td><td>Tyr</td><td>Asn</td><td>Cheese</td><td>Thr</td><td>Tyr 100</td><td>Arg</td><td>Val</td><td></td>

<td>gtc</td><td>age</td><td>gtc</td><td>ctc</td><td>acc</td><td>gtc</td><td>ctg</td><td>cac</td><td>cag</td><td>jacket</td><td>tgg</td><td>ctg</td><td>aat</td><td>ggc</td><td>aag</td><td>gag</td><td> 392</td>

<td>Val</td><td>Cheese</td><td>Val 105</td><td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>His 110</td><td>Gin</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Asn 115</td><td>Gly</td><td>Lys</td><td>Glu</td><td></td>

<td>trays</td><td>aag</td><td>tgc</td><td>aag</td><td>gtc</td><td>tcc</td><td>aac</td><td>aaa</td><td>gee</td><td>ctc</td><td>cca</td><td>gee</td><td>ccc</td><td>atc</td><td>gag</td><td>aaa</td><td> 440</td>

<td>Tyr</td><td>Lys 120</td><td>Cys</td><td>Lys</td><td>Val</td><td>Cheese</td><td>Asn 125</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Ala 130</td><td>Pro</td><td>How much</td><td>Glu</td><td>Lys</td><td></td>

<td>acc</td><td>atc</td><td>tcc</td><td>aaa</td><td>gee</td><td>aaa</td><td>hhg</td><td>cag</td><td>ccc</td><td>ega</td><td>gaa</td><td>cca</td><td>cag</td><td>gtg</td><td>trays</td><td>acc</td><td> 488</td>

<td>Thr 135</td><td>How much</td><td>Cheese</td><td>Lys</td><td>Ala</td><td>Lys 140</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Glu 145</td><td>Pro</td><td>Gin</td><td>Val</td><td>Tyr</td><td>Thr 150</td><td></td>

<td>ctg</td><td>ccc</td><td>cca</td><td>tcc</td><td>cgg</td><td>gen</td><td>gag</td><td>ctg</td><td>acc</td><td>aag</td><td>aac</td><td>cag</td><td>gtc</td><td>age</td><td>ctg</td><td>acc</td><td> 536</td>

<td>Leu</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Arg 155</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Lys 160</td><td>Asn</td><td>Gin</td><td>Val</td><td>Cheese</td><td>Leu 165</td><td>Thr</td><td></td>

<td>tgc</td><td>ctg</td><td>gtc</td><td>aaa</td><td>ggc</td><td>ttc</td><td>tat</td><td>ccc</td><td>age</td><td>jacket</td><td>atc</td><td>gee</td><td>gtg</td><td>gag</td><td>tgg</td><td>gag</td><td> 584</td>

<td>Cys</td><td>Leu</td><td>Val</td><td>Lys 170</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Cheese 175</td><td>Asp</td><td>How much</td><td>Ala</td><td>Val</td><td>Glu 180</td><td>Trp</td><td>Glu</td><td></td>

<td>age</td><td>aat</td><td>hhg</td><td>cag</td><td>ccg</td><td>gag</td><td>aac</td><td>aac</td><td>trays</td><td>aag</td><td>acc</td><td>acg</td><td>cct</td><td>ccc</td><td>gtg</td><td>ctg</td><td> 632</td>

<td>Cheese</td><td>Asn</td><td>Gly 185</td><td>Gin</td><td>Pro</td><td>Glu</td><td>Asn</td><td>Asn 190</td><td>Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro 195</td><td>Pro</td><td>Val</td><td>Leu</td><td></td>

PL 211 164 B1

680

<td>jacket</td><td>tcc</td><td>jacket</td><td>ggc</td><td>tcc</td><td>ttc</td><td>ttc</td><td>ctc</td><td>trays</td><td>age</td><td>aag</td><td>ctc</td><td>acc</td><td>gtg</td><td>jacket</td><td>aag</td>

<td>Asp</td><td>Cheese 200</td><td>Asp</td><td>Gly</td><td>Cheese</td><td>Phe</td><td>Phe 205</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Lys</td><td>Leu 210</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys</td>

<td>age</td><td>agg</td><td>tgg</td><td>cag</td><td>cag</td><td>hhg</td><td>aac</td><td>gtc</td><td>ttc</td><td>tea</td><td>tgc</td><td>tcc</td><td>gtg</td><td>atg</td><td>cat</td><td>gag</td>

<td>Cheese 215</td><td>Arg</td><td>Trp</td><td>Main</td><td>Main</td><td>Gly 220</td><td>Asn</td><td>Val</td><td>Phe</td><td>Cheese</td><td>Cys 225</td><td>Cheese</td><td>Val</td><td>Underworld</td><td>His</td><td>Glu 230</td>

<td>get</td><td>ctg</td><td>cac</td><td>aac</td><td>cac</td><td>trays</td><td>acg</td><td>cag</td><td>aag</td><td>age</td><td>ctc</td><td>tcc</td><td>ctg</td><td>tet</td><td>ccg</td><td>ggt</td>

<td>Ala</td><td>Leu</td><td>His</td><td>Asn</td><td>His 235</td><td>Tyr</td><td>Thr</td><td>Main</td><td>Lys</td><td>Cheese 240</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro 245</td><td>Gly</td>

728

776 aaa taatggatcc Lys

789

<td> &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt;</td><td>12 247 PRT Artificial</td><td>sequence a</td>

<td> &lt;220&gt; &lt;223&gt;</td><td>Construct</td><td>synthetic</td>

<td> &lt;400&gt;</td><td> 12</td><td></td>

<td colspan="2">Met Ile Glu Gly</td><td>Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala Gly</td>

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

70 75 80

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

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 Cheese 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

PL 211 164 B1

<td>Lys</td><td>Leu 210</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys</td><td>Cheese 215</td><td>Arg</td><td>Trp</td><td>Main</td><td>Main</td><td>Gly 220</td><td>Asn</td><td>Val</td><td>Phe</td><td>Cheese</td>

<td>Cys</td><td>Cheese</td><td>Val</td><td>Underworld</td><td>His</td><td>Glu</td><td>Ala</td><td>Leu</td><td>His</td><td>Asn</td><td>His</td><td>Tyr</td><td>Thr</td><td>Main</td><td>Lys</td><td>Cheese</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Lys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

245 &lt;210&gt; 13 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; ΤΜΡ &lt;400&gt; 13

Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu Ala Ala Arg Ala 15 10

<td> &lt;210&gt;</td><td> 14</td>

<td> &lt;211&gt;</td><td> 36</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Artificial</td>

<td> &lt;220&gt;</td><td></td>

<td> &lt;223&gt;</td><td>TMP-TMP</td>

<td> &lt;400&gt;</td><td> 14</td>

<td colspan="2">How much Glu Gly Pro</td>

Gly Gly Gly Gly Gly Gly Ile Glu Gly Pro Thr Leu Arg Gln Trp Leu 20 25 30

Ala Ala Arg Ala 35 &lt;210&gt; 15 &lt;211&gt; 812 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; EMP-Fc &lt;220&gt;

&lt;221&gt; CDS &lt;222&gt; (39) .. (797) &lt;400&gt; 15 tctagatttg ttttaactaa ttaaaggagg aataacat atg gac aaa act cac aca Met Asp Lys Thr His Thr 1 5 tgt cca cct tgt cca gct ccg gaa ctc ctc ggg gga ctc tca gtc ttc Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

15 20

104

PL 211 164 B1

<td>ctc Leu</td><td>ttc Phe</td><td>ccc Pro 25</td><td>cca Pro</td><td>aaa Lys</td><td>ccc Pro</td><td>aag Lys</td><td>jacket Asp thirty</td><td>acc Thr</td><td>ctc Leu</td><td>atg Underworld</td><td>atc How much</td><td>tcc Cheese 35</td><td>cgg Arg</td><td>acc Thr</td><td>cct Pro</td><td> 152</td>

<td>gag</td><td>gte</td><td>aca</td><td>tgc</td><td>gtg</td><td>gtg</td><td>gtg</td><td>jacket</td><td>gtg</td><td>agc</td><td>cac</td><td>gaa</td><td>jacket</td><td>cct</td><td>gag</td><td>gte</td><td> 200</td>

<td>Glu</td><td>Val</td><td>Thr</td><td>Cys</td><td>Val</td><td>Val</td><td>Val</td><td>Asp</td><td>Val</td><td>Cheese</td><td>His</td><td>Glu</td><td>Asp</td><td>Pro</td><td>Glu</td><td>Val</td><td></td>

<td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td><td> 50</td><td></td><td></td><td></td><td></td><td></td>

<td>aag</td><td>ttc</td><td>aac</td><td>tgg</td><td>trays</td><td>gtg</td><td>jacket</td><td>ggc</td><td>gtg</td><td>gag</td><td>gtg</td><td>cat</td><td>aat</td><td>gcc</td><td>aag</td><td>aca</td><td> 248</td>

<td>Lys</td><td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Gly</td><td>vai</td><td>Glu</td><td>Val</td><td>His</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Thr</td><td></td>

<td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td>

<td>aag</td><td>ccg</td><td>cgg</td><td>gag</td><td>gag</td><td>cag</td><td>trays</td><td>aac</td><td>agc</td><td>acg</td><td>trays</td><td>cgt</td><td>gtg</td><td>gte</td><td>agc</td><td>gte</td><td> 296</td>

<td>Lys</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Tyr</td><td>Asn</td><td>Cheese</td><td>Thr</td><td>Tyr</td><td>Arg</td><td>Val</td><td>Val</td><td>Cheese</td><td>Val</td><td></td>

<td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td>

<td>ctc</td><td>acc</td><td>gte</td><td>ctg</td><td>cac</td><td>cag</td><td>jacket</td><td>tgg</td><td>ctg</td><td>aat</td><td>ggc</td><td>aag</td><td>gag</td><td>trays</td><td>aag</td><td>tgc</td><td> 344</td>

<td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>His</td><td>Gin</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Asn</td><td>Gly</td><td>Lys</td><td>Glu</td><td>Tyr</td><td>Lys</td><td>Cys</td><td></td>

<td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td>

<td>aag</td><td>gte</td><td>tcc</td><td>aac</td><td>aaa</td><td>gcc</td><td>ctc</td><td>cca</td><td>gcc</td><td>ccc</td><td>atc</td><td>gag</td><td>aaa</td><td>acc</td><td>atc</td><td>tcc</td><td> 392</td>

<td>Lys</td><td>Val</td><td>Cheese</td><td>Asn</td><td>Lys</td><td>Ala</td><td>Leu</td><td>Pro</td><td>Ala</td><td>Pro</td><td>How much</td><td>Glu</td><td>Lys</td><td>Thr</td><td>How much</td><td>Cheese</td><td></td>

<td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td>

<td>aaa</td><td>gcc</td><td>aaa</td><td>hhg</td><td>cag</td><td>ccc</td><td>ega</td><td>gaa</td><td>cca</td><td>cag</td><td>gtg</td><td>trays</td><td>acc</td><td>ctg</td><td>ccc</td><td>cca</td><td> 440</td>

<td>Lys</td><td>Ala</td><td>Lys</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Pro</td><td>Gin</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td><td></td>

<td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td><td> 130</td><td></td><td></td><td></td><td></td><td></td>

<td>tcc</td><td>cgg</td><td>gen</td><td>gag</td><td>ctg</td><td>acc</td><td>aag</td><td>aac</td><td>cag</td><td>gte</td><td>agc</td><td>ctg</td><td>acc</td><td>tgc</td><td>ctg</td><td>gte</td><td> 488</td>

<td>Cheese</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Thr</td><td>Lys</td><td>Asn</td><td>Gin</td><td>Val</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Cys</td><td>Leu</td><td>Val</td><td></td>

<td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td>

<td>aaa</td><td>ggc</td><td>ttc</td><td>tat</td><td>ccc</td><td>agc</td><td>jacket</td><td>atc</td><td>gcc</td><td>gtg</td><td>gag</td><td>tgg</td><td>gag</td><td>agc</td><td>aat</td><td>hhg</td><td> 536</td>

<td>Lys</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>Asp</td><td>How much</td><td>Ala</td><td>Val</td><td>Glu</td><td>Trp</td><td>Glu</td><td>Cheese</td><td>Asn</td><td>Gly</td><td></td>

<td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td>

<td>cag</td><td>ccg</td><td>gag</td><td>aac</td><td>aac</td><td>trays</td><td>aag</td><td>acc</td><td>acg</td><td>cct</td><td>ccc</td><td>gtg</td><td>ctg</td><td>jacket</td><td>tcc</td><td>jacket</td><td> 584</td>

<td>Gin</td><td>Pro</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>Lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>Val</td><td>Leu</td><td>Asp</td><td>Cheese</td><td>Asp</td><td></td>

<td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td>

<td>ggc</td><td>tcc</td><td>ttc</td><td>ttc</td><td>ctc</td><td>trays</td><td>agc</td><td>aag</td><td>ctc</td><td>acc</td><td>gtg</td><td>jacket</td><td>aag</td><td>agc</td><td>agg</td><td>tgg</td><td> 632</td>

<td>Gly</td><td>Cheese</td><td>Phe</td><td>Phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>Lys</td><td>Leu</td><td>Thr</td><td>Val</td><td>Asp</td><td>Lys</td><td>Cheese</td><td>Arg</td><td>Trp</td><td></td>

<td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td>

<td>cag</td><td>cag</td><td>hhg</td><td>aac</td><td>gte</td><td>ttc</td><td>tca</td><td>tgc</td><td>tcc</td><td>gtg</td><td>atg</td><td>cat</td><td>gag</td><td>gct</td><td>ctg</td><td>cac</td><td> 680</td>

<td>Gin</td><td>Gin</td><td>Gly</td><td>Asn</td><td>Val</td><td>Phe</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>Val</td><td>Underworld</td><td>His</td><td>Glu</td><td>Ala</td><td>Leu</td><td>His</td><td></td>

<td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td><td> 210</td><td></td><td></td><td></td><td></td><td></td>

<td>aac</td><td>cac</td><td>trays</td><td>acg</td><td>cag</td><td>aag</td><td>agc</td><td>ctc</td><td>tcc</td><td>ctg</td><td>tct</td><td>ccg</td><td>ggt</td><td>aaa</td><td>ggt</td><td>gga</td><td> 728</td>

<td>Asn</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>Lys</td><td>Gly</td><td>Gly</td><td></td>

<td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td>

<td>ggt</td><td>ggt</td><td>ggt</td><td>gga</td><td>ggt</td><td>act</td><td>trays</td><td>tct</td><td>tgc</td><td>cac</td><td>ttc</td><td>ggc</td><td>ccg</td><td>ctg</td><td>act</td><td>tgg</td><td> 776</td>

<td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Thr</td><td>Tyr</td><td>Cheese</td><td>Cys</td><td>His</td><td>Phe</td><td>Gly</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Trp</td><td></td>

<td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td>

<td>gtt</td><td>tgc</td><td>aaa</td><td>ccg</td><td>cag</td><td>ggt</td><td>ggt</td><td colspan="3">taatctcgtg &lt;</td><td colspan="2">gatcc</td><td></td><td></td><td></td><td></td><td> 812</td>

<td>Val</td><td>Cys</td><td>Lys</td><td>Pro</td><td>Gin</td><td>Gly</td><td>Gly</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 16 &lt;211&gt; 253

250

&lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; synthetic construct &lt;400&gt; 16

Met Asp Lys Thr His Thr Cys Pro Pro 1 5

Cys Pro Ala Pro Glu Leu 10 15

Gly Gly Pro Ser Val Phe Leu Phe Pro 20 25

Pro Lys Pro Lys Asp Thr 30

Met Ile Ser Arg Thr Pro Glu Val Thr 35 40

Cys Val Val Val Asp Val 45

His Glu Asp Pro Glu Val Lys Phe Asn 50 55

Trp Tyr Val Asp Gly Val 60

Val His Asn Ala Lys Thr Lys Pro Arg 65 70

Glu Glu Gln Tyr Asn Ser 75

Tyr Arg Val Val Ser Val Leu Thr Val 85

Leu His Gln Asp Trp Leu 90 95

Gly Lys Glu Tyr Lys Cys Lys Val Ser 100 105

Asn Lys Ala Leu Pro Ala 110

Ile Glu Lys Thr Ile Ser Lys Ala Lys 115 120

Gly Gln Pro Arg Glu Pro 125

Val Tyr Thr Leu Pro Pro Ser Arg Asp 130 135

Glu Leu Thr Lys Asn Gln 140

Leu Thr Cys Leu Val Lys Gly Phe Cheese 145 150

Tyr Pro Ser Asp Ile Ala 155

Glu Trp Glu Cheese Asn Gly Gln Pro Glu 165

Asn Asn Tyr Lys Thr Thr 170 175

Pro Val Leu Asp Ser Asp Gly Ser Phe 180 185

Phe Leu Tyr Ser Lys Leu 190

Val Asp Lys Ser Arg Trp Gln Gln Gly 195 200

Asn Val Phe Ser Cys Ser 205

Met His Glu Ala Leu His Asn His Tyr 210 215

Thr Gln Lys Ser Leu Ser 220

Pro Gly Lys Gly Gly Gly Gly Gly Cheese 225 230

Gly Gly Thr Tyr Ser Cys 235

Leu

Leu

Cheese

Glu

Thr

Asn

Pro

Main

Val

Val

160

Pro

Thr

Val

Leu

His

240

Phe Gly Pro Leu Thr Trp Val Cys Lys 245

Pro Gln Gly Gly 250