AU2004200691B2 - Modified peptides as therapeutic agents - Google Patents

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant: Actual Inventors: Address for Service:

CCN:

Amgen Inc.

Ulrich Feige and Chuan-Fa Liu and Janet C. Cheetham and Thomas Charles. Boone Baldwin Shelston Waters MARGARET STREET SYDNEY NSW 2000 3710000352 Invention Title: MODIFIED PEPTIDES AS THERAPEUTIC AGENTS Details of Original Application No. 12322/00 dated 25 October 1999 The following statement is a full description of this invention, including the best method of performing it known to us:- File: 31352AUP01 500308409_1.DOC/5844 Modified Peptides as Therapeutic Agents Background of the Invention Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemical modification. Such modifications can protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Protein modifications may also increase the therapeutic protein's stability, circulation time, and biological activity. A review article describing protein modification and fusion protein is Francis (1-992), Focus on Growth Factors 3:4-10 (Mediscript, London), which is hereby incorporated by reference.

One useful modification is combination with the "Fc" domain of an antibody.

Antibodies comprise two functionally independent parts, a variable domain known as "Fab", which binds antigen, and a constant domain known as which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989), Nature 337:525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusions known in the art.

-2- Table 1-Fc fusion with therapeutic proteins Form of Fc Fusion Therapeutic partner implications Reference IgG1 N-terminus of Hodgkin's disease; U.S. Patent No.

anaplastic lymphoma; T- 5,480,981 cell leukemia Murine Fcy2a IL-10 anti-inflammatory; Zheng tal. (1995), L transplant rejection Immunol. 154:5590-600 IgG1 TNF receptor septic shock Fisher eal. (1996), N.

Engl. J. Med. 334:1697- 1702; Van Zee, K. eLaL (1996), J. Immunol. 156: 2221-30 IgG, IgA, TNF receptor inflammation, autoimmune U.S. Pat. No. 5,808,029, IgM, or IgE disorders issued September (excluding 1998 the first domain) IgG1 CD4 receptor AIDS Capon eLal (1989), Natre 337: 525-31 IgG1, N-terminus anti-cancer, antiviral Harvill etLa (1995), lgG3 of IL-2 Immunotech. 1:95-105 IgG1 C-terminus of osteoarthritis; WO 97/23614, published OPG bone density July 3, 1997 IgG1 N-terminus of anti-obesity PCT/US 97/23183, filed leptin December 11, 1997 Human Ig CTLA-4 autoimmune disorders Linsley (1991), J.ExD.

Cy1 Med. 174:561-9 A much different approach to development of therapeutic agents is peptide library screening. The interaction of a protein ligand with its receptor often takes place at a relatively large interface. However, as demonstrated for human growth hormone and its receptor, only a few key residues at the interface co-nitibute to most of the binding energy.

Clackson et al. (1995), Science 267:383-6. The bulk of the protein ligand merely displays the binding epitopes in the right topology or serves functions unrelated to binding. Thus, molecules of only "peptide" length (2 to 40 amino acids) can bind to the receptor protein of a given large protein ligand. Such peptides may mimic the bioactivit of the-large protein ligand ("peptide agonists") or, through competitive binding, inhibit the bioactivity of the large protein ligand ("peptide antagonists").

-3- Phage display peptide libraries have emerged as a powerful method in identifying such peptide agonists and antagonists. See, for example, Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued June 29, 1993; U.S. Pat. No.

5,733,731, issued March 31,1998; U.S. Pat. No. 5,498,530, issued March 12, 1996; U.S. Pat. No. 5,432,018, issued July 11, 1995; U.S. Pat. No. 5,338,665, issued August 16, 1994; U.S. Pat. No. 5,922,545, issued July 13, 1999; WO 96/40987, published December 19, 1996; and WO 98/15833, published April 16, 1998 (each of which is incorporated by reference). In such libraries, random peptide sequences are displayed by fusion with coat proteins of filamentous phage. Typically, the displayed peptides are affinity-eluted against an antibody-immobilized extracellular domain of a receptor. The retained phages may be enriched by successive rounds of affinity purification and repropagation. The best binding peptides may be sequenced to identify key residues within one or more structurally related families of peptides. See, Cwirla et al. (1997), Science 276: 1696-9, in which two distinct families were identified. The peptide sequences may also suggest which residues may be safely replaced by alanine scanning or by mutagenesis at the DNA level. Mutagenesis libraries may be created and screened to further optimize the sequence of the best binders.

Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26:401-24.

Structural analysis of protein-protein interaction may also be used to suggest peptides that mimic the binding activity of large protein ligands. In such an analysis, the crystal structure may suggest the identity and relative orientation of critical residues of the large protein ligand, from which a peptide may be designed. See, Takasaki et al. (1997), Nature Biotech. 15: 1266-70. These analytical methods may-also-be used to investigate the interaction between a receptor protein and peptides

I

selected by phage display, which may suggest further modification of the peptides to increase binding affinity.

Other methods compete with phage display in peptide research. A peptide library can be fused to the carboxyl terminus of the lac.repressor and expressed in E. coli. Another E. coli-based method allows display on the cell's outer membrane by fusion with a peptidoglycan-associated lipoprotein (PAL). Hereinafter, these and related methods are collectively referred to as coli display." In another method, translation of random RNA is halted prior to ribosome release, resulting in a library of polypeptides with their associated RNA still attached. Hereinafter, this and related methods are collectively referred to as "ribosome display." Other methods employ chemical linkage of peptides to RNA; see, for example, Roberts Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297- 303. Hereinafter, this and related methods are collectively referred to as "RNA-peptide screening." Chemically derived peptide libraries have been developed in which peptides are immobilized on stable, non-biological materials, such as polyethylene rods or solvent-permeable resins. Another chemically derived peptide library uses photolithography to scan peptides immobilized on glass slides. Hereinafter, these and related methods are collectively referred to as "chemical-peptide screening." Chemical-peptide screening may be advantageous in that it allows use of D-amino acids and other unnatural analogues, as well as non-peptide elements. Both biological and chemical methods are reviewed in Wells Lowman (1992), Curr. Opin. Biotechnol. 3:355-62.

Conceptually, one may discover peptide mimetics of any protein using phage display and the other methods mentioned above. These methods have been used for epitope mapping, for identification of critical amino acids in protein-protein interactions, and as leads for the discovery of new therapeutic agents. Cortese et al. (1996), Curr. Opin. Biotech. 7: 616-21. Peptide libraries are now being used most often in immunological studies, such as epitope mapping. Kreeger (1996), The Scientist 10(13): 19- Of particular interest here is use of peptide libraries and other techniques in the discovery of pharmacologically active peptides. A number of such peptides identified in the art are summarized in Table 2.

The peptides are described in the listed publications, each of which is hereby incorporated by reference. The pharmacologic activity of the peptides is described, and in many instances is followed by a shorthand term therefor in parentheses. Some of these peptides have been modified to form C-terminally cross-linked dimers). Typically, peptide libraries were screened for binding to a receptor for a pharmacologically active protein EPO receptor). In at least one instance (CTLA4), the peptide library was screened for binding to a monclonal antibody.

I

Table 2-Pharmacologically active peptides Form of peptide Binding partner/ protein of i rterest Pharmacologic activity Reference intrapeptide EPO receptor EPO-mimetic Wrighton eti. (1996), disulfide Science 273: 458-63; bonded U.S. Pat. No. 5,773,569, issued June 30, 1998 to Wriqhton eal.

C-terminally EPO receptor EPO-mimetic Livnah (1996), cross-linked iene 273: 464-71; dimer Wrighton f l.(1997), mrNature Biotechnoloav 1261-5; International patent application

WO

96/40772, published Dec. 19, 1996 linear EPO receptor EPO-mimetic Narandaeta. (1999), Proc. Natl. Acad. Sci.

USA. 96: 7569-74 linear c-Mpl TPO-mimetic Cwirla tULa.(1997) Sciene 276: 1696-9; U.S. Pat. No. 5,869,451, issued Feb. 9,1999; U.S.

Pat. No. 5,932,946, issued Aug. 3,1999 C-terminally c-Mpl TPO-mimetic Cwirla (1997), cross-linked Sciene 276:1696-9 dimer disulfide- stimulation of Paukovits al. (1984), linked dimer hematopoiesis Hppe-Sevles

Z.

("G-CSF-mimetic") Physiol. Chem. 365: 303- 11; Laerum etal. (1988), EX Hemat. 16:274-80 alkylene- G-CSF-mimetic Bhatnagar Lal. (1996), lkylend J. Med. Chem. 39: 3814linked dimer 9; Cuthbertson tal.

(1997), J. Med. Chem.

2876-82; King etal.

(1991), Ex. Hematol.

19:481; King etal.

(1995), Blood 86 (Suppl.

309a linear IL-1 receptor inflammatory and U.S. Pat. No. 5,608,035; autoimmune diseases U.S. Pat. No. 5,786,331; ("IL-1 antagonist" or U.S-Pat. No. 5,880,096; "IL-1 ra-mimetic") Yanofsky etal. (1996), "The protein listed in this column may be bound by the associated peptide

EPO

receptor, IL-1 receptor) or mimicked by the associated peptide. The references listed for each clarify whether the molecule is bound by or mimicked by the peptides.

-7- Pro. Nti Acd-.9.i.93: 7381 Akeson &tal.

271: 30517-23; Wiekzorek aL1 (1997),.

pcI. J. Parmacol. 49: 1 07-17;-YalofSkY (1996), PNAs, 93:7381-7386.

linear Facteur stimulation of Indgaki-Ohara thymnique lymphocytes (1996), Caiidlflu P31mmuno serique (FTS) C"FTS-mimetiC") 171: 30-40; Yoshida (1984), inL~L IMMvunOahrmACD1 6:141-6.

intrapeptide CTLA4 MAE CLA4-rnimetic Fukumoto eLaL (1998), disulfideNature ioteh. 16: 267bonded 70aak (19) exocyclic TNF-cz receptor TNF-cz antagonist Takaak LtLai. (197) Nature Bioteh. 15:1 266- WO 98/53842, published December 3, 1998 linear TN F-cz receptor. TNF-aL antagonist Chirinos-Rojas(),L Imm.n, 5621-5626.

intrapeptide 03b hinhibition of complemeint Sahu (1996), 1L disulfide activation; autoiMmune Immunal. 157:884-91; bonded diseases -Morikis "La. (1998), (1"c3b-antagoflIt") _Prot ein ci. 7: 619-2 lin ear vinculin cell adhesion processes- Adey ;ZI. (1 997), cell growth, differentiation, %=hem.ILJ. 324: 523-8 wound healing, tumor metastasis ("vinculin binding'I linear 04 binding anti-thromb:)tiC Linse W.Lai. (1997),

J,

protein~5. (CB) iL 272:14658protin P13P)65 linear urokinase processes associated with Goodson eL-al. (1994), receptor urokinaSe interaction with ProQN 4ILdA=~I 91 its receptor 7129-33; International angiogenesis, tumor cell application

WO

invasion and metastasis); 97/35969, published ("UKR anta gonist") October 2, 1997 lier Mdm2, Inh Ii ibition of inactivation of Picley lia 1. (1 994), linear p53 mediated by Mdm2 or _QnggnDq 9: 2523-9; hdm2; anti-tumor Bottger eLz1. (1997) 1L ("Mdm/hdm antagonist') hMgLBiQi. 269: 744-56; Bottger gLa1.(i1 996 WAr 1 anti-tum~lor by mimicking _BliL (1997), Curr.

the activity1 of p)21 Fl BQio. 7: 71-80 linear farnesyl anti-cancer by Preventing Gibbs et al. (1994), Cell b FTS is a thymic hormone mimicked by the molecule of this invention rather than a receptor bound by the molecule of this invention.

transferase activation of ras oncogene 77:175-178 linear Ras effector anti-cancer by inhibiting Moodie et al. (1994), domain biological function of the Trends Genet 10: 44-48 ras oncogene Rodriguez et al. (1994), Nature 370:527-532 linear SH2/SH3 anti-cancer by inhibiting Pawson et al (1993), domains tumor growth with Curr. Biol. 3:434-432 activated tyrosine kinases Yu et al. (1994), Cell 76:933-945 linear p16'NK4 anti-cancer by mimicking FAhraeus al. (1996), activity of p16; Curr. Biol. 6:84-91 inhibiting cyclin D-Cdk complex ("p16-mimetic") linear Src, Lyn inhibition of Mast cell Stauffer Lal. (1997), activation, IgE-related Biochem. 36: 9388-94 conditions, type I hypersensitivity ("Mast cell antagonist") linear Mast cell treatment of inflammatory International application protease disorders mediated by WO 98/33812, published release of tryptase-6 August 6, 1998 ("Mast cell protease inhibitors") linear SH3 domains treatment of SH3- Rickles Laet. (1994), mediated disease states EMB J. 13: 5598-5604; ("SH3 antagonist") Sparks 'al. (1994), JL Biol.Chem. 269: 23853- 6; Sparks eal. (1996), Proc. Natl. Acad. Sci. 93: 1540-4 linear HBV core treatment of HBV viral Dyson Muray (1995), antigen (HBcAg) infections ("anti-HBV") Proc. Natl. Acad. Sci. 92: 2194-8 linear selectins neutrophiladhesion; Martens tal. (1995), L inflammatory diseases Biol. ChM. 270: 21129- ("selectin antagonist") 36; European patent application EP 0 714 912, published June 1996 linear, calmodulin calmodulin antagonist Pierce tal. (1995), cyclized Molec. Dversty 1: 259c ycl ze d 65; Dedman elal.

(1993), J. Bil. Chem.

268:23025-30; Adey Kay (1996), Qn 169: 133-4 linear, integrins tumor-homing; treatment International applications cyclized- for conditions related to WO 95/14714, published integrin-mediated cellular Juneu 1, 1995; WO events, including platelet 97/08203, published aggregation, thrombosis, March 6, 1997; WO wound healing, 98/10795, published osteoporosis, tissue March 19, 1998; WO repair, angiogenesis 99/24462, published May -9for treatment of cancer), 20, 1999; Kraft tal.

and tumor invasion (1999), J. Biol. Chem.

("integrin-binding") 274:1979-1985 cyclic, linear fibronectin and treatment of inflammatory WO 98/09985, published extracellular and autoimmune March 12,1998 matrix conditions components of T cells and macrophages linear somatostatin treatment or prevention of European patent and cortistatin hormone-producing application 0 911393, tumors, acromegaly, published April 28, 1999 giantism, dementia, gastric ulcer, tumor growth, inhibition of hormone secretion, modulation of sleep or neural activity linear bacterial antibiotic; septic shock; U.S. Pat. No. 5,877,151, lipopolySac- disorders modulatable by issued March 2, 1999 charide CAP37 linear or pardaxin, mellitin antipathogeic W 97/3119, published cyclic, 28 August 1997 cyclic, including Damino acidsr, cyclic VP impotence, WO 97/40070, published linear, cyclic neurodegenerative October 30, 1997 disorders linear CTLs cancer EP 0 770 624, published May 2, 1997 linear THF-gamma2 Burnstein (1988), linear THF-gamma2 he., 27:4066-71.

Cooper (1987), Proc.

linear Amylin Natl Acad. ScL 84:8628-32.

linear Adrenomedullin 192:55Kitamura (1993), -60 cyclic, linear VEGF anti-angiogenic; cancer, Fairbrother (1998), rheumatoid arthritis, Biochem., 37:17754diabetic retinopathy, 17764.

psoriasis

("VEGF

antagonist") cyclic MMP inflammation and Koivunen (1999), Nature autoimmune disorders; Bigech., 17:768-774.

tumor growth ("MMP inhibitor") HGH fragme U.S. Pat. No. 5,869,452 Echistatin inhibition of platelet Gan (1988), J. BL a gregation Qbl I, 263:19827-32.

*linear SLE SLE WO 96/30057, published autoantblinear SLE October 3, 1996 GD1 alpha suppression of tumor Ishikawa nb. (1998), metastasis FE La-tt 441 20-4 antiphosphoipi endothelial cell activation, lank ta. (1999), Pr beta-2- antiphospholipid Natl. Acad. Sci. USA glycoprotein-1 syndrome (APS), 96: 5164-8 (p2GPI) thromboembolic antibodies phenomena, thrombocytopenia, and recurrent fetal loss linear T Cell diabetes WO 96/11214, Receptor published April 18, beta chain 1996 Peptides identified by peptide library screening have been regarded as "leads" in development of therapeutic agents rather than as therapeutic agents themselves. Like other proteins and peptides, they would be rapidly removed in vivo either by renal filtration, cellular clearance mechanisms in the reticuloendothelial system, or proteolytic degradation. Francis (1992), Focus on Growth Factors 3: 4-11. As a result, the art presently uses the identified peptides to validate drug targets or as scaffolds for design of organic compounds that might not have been as easily or as quickly identified through chemical library screening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24; Kay et o0 al. (1998), Drug Disc. Today 3: 370-8. The art would benefit from a process by which such peptides could more readily yield therapeutic agents.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Summary of the Invention The present invention concerns a process by which the in vivo half-life of one or more biologically active peptides is increased by fusion with a vehicle. In this invention, pharmacologically active compounds are prepared by a process comprising: a) selecting at least one peptide that modulates the activity of a protein of interest; and b) preparing a pharmacologic agent comprising at least one vehicle lOa- Scovalently linked to at least one amino acid sequence of the selected cpeptide.

SAccording to a first aspect of the invention there is provided a composition of matter of the formula 2 )b 0and multimers thereof, wherein: O F 1 is an Fc domain;

X

1 and X 2 are each independently selected from 1 1

(L

2 )d _P2, -(L1)c-P (L 2 )d_p2_(L 3 )e-P 3 and 2 )d-P 2

-(L

3 )e -P 3

-(L

4 )pP 4 PI, P2, P3, and P4 are each independently randomized GCSF binding peptide sequences;

L

1

L

2

L

3 and L 4 are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1 and wherein "peptide" refers to molecules 2 to 40 amino acids and wherein X 1 nor X 2 is a native protein.

According to a second aspect of the invention there is provided a DNA encoding a composition of matter according to the first aspect.

According to a third aspect of the invention there is provided an expression vector comprising the DNA of the second aspect.

According to a fourth aspect of the invention there is provided a host cell comprising the expression vector of the third aspect.

According to a fifth aspect of the invention there is provided a process for preparing a GCSF binding compound, which comprises a) selecting at least one randomized non-native GCSF-binding peptide of 2 to 40 amino acids in length; and b) preparing a GCSF binding compound comprising at least one Fc domain covalently linked to at least one amino acid sequence of the selected peptide or peptides.

The preferred vehicle is an Fc domain. The peptides screened in step are preferably expressed in a phage display library. The vehicle and the -11peptide may be linked through the N- or C-terminus of the peptide or the vehicle, as described further below. Derivatives of the above compounds (described below) are also encompassed by this invention.

The compounds of this invention may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins. Compounds of this invention that encompass non-peptide portions may be synthesized by standard organic chemistry reactions, in addition to standard peptide chemistry reactions when applicable.

The primary use contemplated is as therapeutic or prophylactic agents. The vehicle-linked peptide may have activity comparable to-or even greater than-the natural ligand mimicked by the peptide. In addition, certain natural ligand-based therapeutic agents might induce antibodies against the patient's own endogenous ligand; the vehicle-linked peptide avoids this pitfall by having little or typically no sequence identity with the natural ligand.

Although mostly contemplated as therapeutic agents, compounds of this invention may also be useful in screening for such agents. For example, one could use an Fc-peptide Fc-SH2 domain peptide) in an 2 0 assay employing anti-Fc coated plates. The vehicle, especially Fc, may make insoluble peptides soluble and thus useful in a number of assays.

The compounds of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other mammal) in need thereof. Other related aspects are also included in the instant invention.

Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.

-12- Brief Description of the Figures Figure 1 shows a schematic representation of an exemplary process of the invention. In this preferred process, the vehicle is an Fc domain, which is linked to the peptide covalently by expression from a DNA construct encoding both the Fc domain and the peptide. As noted in Figure 1, the Fc domains spontaneously form a dimer in this process.

Figure 2 shows exemplary Fc dimers that may be derived from an IgG1 antibody. "Fc" in the figure represents any of the Fc variants within the meaning of "Fc domain" herein. and represent peptides or linker-peptide combinations as defined hereinafter. The specific dimers are as follows: A, D: Single disulfide-bonded dimers. IgG1 antibodies typically have two disulfide bonds at the hinge region between the constant and variable domains. The Fc domain in Figures 2A and 2 D may be formed by truncation between the two disulfide bond sites or by substitution of a cysteinyl residue with an unreactive residue alanyl). In Figure 2A, the Fc domain is linked at the amino terminus of the peptides; in 2D, at the carboxyl terminus.

B, E: Doubly disulfide-bonded dimers. This Fc domain may be formed by truncation of the parent antibody to retain both cysteinyl residues in the Fc domain chains or by expression from a construct including a sequence encoding such an Fc domain. In Figure 2B, the Fc domain is linked at the amino terminus of the peptides; in 2E, at the carboxyl terminus.

C, F: Noncovalent dimers. This Fc domain may be formed by elimination of the cysteinyl residues by either truncation or substitution.

One may desire to eliminate the cysteinyl residues to avoid impurities formed by reaction of the cysteinyl residue with cysteinyl residues of other -13proteins present in the host cell. The noncovalent bonding of the Fc domains is sufficient to hold together the dimer.

Other dimers may be formed by using Fc domains derived from different types of antibodies IgG2, IgM).

Figure 3 shows the structure of preferred compounds of the invention that feature tandem repeats of the pharmacologically active peptide. Figure 3A shows a single chain molecule and may also represent the DNA construct for the molecule. Figure 3B shows a dimer in which the linker-peptide portion is present on only one chain of the dimer.

Figure 3C shows a dimer having a peptide portion on both chains. The dimer of Figure 3C will form spontaneously in certain host cells upon expression of a DNA construct encoding the single chain shown in Figure 3A. In other host cells, the cells could be placed in conditions favoring formation of dimers or the dimers can be formed in vitro.

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

Figure 5 shows a synthetic scheme for the preparation ofPEGylated peptide 19 (SEQ ID NO: 3) as prepared through intermediates having SEQ ID NOS:1128 through 1131, respectively.

Figure 6 shows a synthetic scheme for the preparation of PEGylated peptide (SEQ ID NO: 4) as prepared through intermediates having SEQ ID NOS: 1132 and 1133, respectively.

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 hereinafter.

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 hereinafter.

-14- 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 hereinafter.

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 hereinafter.

Figure 11 shows the number of platelets generated in vivo in normal female BDF1 mice treated with one 100 4g/kg bolus injection of various compounds, with the terms defined as follows. PEG-MGDF: 20 kD average molecular weight PEG attached by reductive amination to the N-terminal amino group of amino acids 1-163 of native human TPO, which is expressed in E. coli (so that it is not glycosylated); TMP: the TPO-mimetic peptide having the amino acid sequence IEGPTLRQWLAARA (SEQ ID NO: 13); TMP-TMP: the TPO-mimetic peptide having the amino acid sequence IEGPTLRQWLAARA-GGGGGGG- IEGPTLRQWLAARA (SEQ ID NO: 14); PEG-TMP-TMP: the peptide of SEQ ID NO: 14, wherein the PEG 2 0 group is a 5 kD average molecular weight PEG attached as shown in Figure 6; Fc-TMP-TMP: the compound of SEQ ID NO: 8 (Figure 8) dimerized with an identical second monomer Cys residues 7 and are bound to the corresponding Cys residues in the second monomer to form a dimer, as shown in Figure and TMP-TMP-Fc is the compound of SEQ ID NO: 10 (Figure 9) dimerized in the same way as TMP-TMP-Fc except that the Fc domain is attached at the C-terminal end rather than the Nterminal end of the TMP-TMP peptide.

Figure 12 shows the number of platelets generated in vivo in normal BDF1 mice treated with various compounds delivered via implanted osmotic pumps over a 7-day period. The compounds are as defined for 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 hereinafter.

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 hereinafter.

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 hereinafter.

Figure 16 shows the nucleotide and amino acid sequences (SEQ

ID

NOS: 21 and 22, respectively) of the molecule identified as "Fc-EMP-EMP" inExample 3 hereinafter.

Figures 17A and 17B show the DNA sequence (SEQ ID NO: 23) inserted into pCFM1656 between the unique AatlI (position #4364 in pCFM165 6 and Sacl (position #4585 in pCFM1656) restriction sites to form expression plasmid pAMG21 (ATCC accession no. 98113).

Figure 18A shows the hemoglobin, red blood cells, and hematocrit generated in vivo in normal female BDF1 mice treated with one 100 pg/kg bolus injection of various compounds. Figure 18B shows the same results with mice treated with 100 pg/kg per day delivered e by 7day micro-osmotic pump with the EMPs delivered at 100 g/kg, rhEPO at (In both experiments, neutrophils, lymphocytes, and platelets were unaffected.) In these figures, the terms are defined as follows.

Fc-EMP: the compound of SEQ ID NO: 16 (Figure 13) dimerized with an identical second monomer Cys residues 7 and 10 are -16bound to the corresponding Cys residues in the second monomer to form a dimer, as shown in Figure 2); EMP-Fc: the compound of SEQ ID NO: 18 (Figure 14) dimerized in the same way as Fc-EMP except that the Fc domain is attached at the C-terminal end rather than the N-terminal end of the EMP peptide.

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

SFigures 19A and 19B show the nucleotide and amino acid sequences (SEQ ID NOS: 1055 and 1056) of the Fc-TNF-a inhibitor fusion molecule described in Example 4 hereinafter.

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

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

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

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

-17- Figures 24A and 24B show the nucleotide and amino acid sequences (SEQ ID NOS: 1065 and 1066) of the VEGF antagonist-Fc fusionmolecule described in Example 6 hereinafter.

Figures 25A and 25B show the nucleotide and amino acid sequences (SEQ ID NOS: 1067 and 1068) o the F-MMP inhibitor fusion molecule described in Example 7 hereinafter.

d esribed in Exand 26B show the nucleotide and amino acid sequences (SEQ ID NOS: 1069 and 1070) of the MMP inhibitor-Fe fusion molecule described in Example 7 hereinafter.

Detailed Description of the Invention Definition of Terms The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.

The term "compising" means that a compound may include additional amino acids on either or both of the N- or C- termini of the given sequence. Of course, these additional amino acids should not significantly interfere with the activity of the compouents d egradation The term "vehicle" refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or 2 0 increases biological activity of a therapeutic protein. Exemplary vehicles include an Fc domain (which is preferred) as well as a linear polymer polyethylene glycol (PEG), polylysine, dextran, a branched-chain polymer (slee p atent No. 4,289,872 to Denkenwalter et al., issued eptember 15,1981; 5,229,490 to Tam, issued july 20,1993; WO 93/21259by Freche t etal., published 28 October 1993); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oigosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor. Vehicles are further described hereinafter.

-18- The term "native Fc" refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class IgG, IgA, IgE) or subclass IgG1, IgG2, IgG3, IgAl, IgGA2). One example of a native Fc is a disulfidebonded dimer resulting from papain digestion of an IgG (see Ellison et al.

(1982), Nucleic Acids Res. 10: 4071-9). The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.

The term "Fc variant" refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. International applications WO 97/34631 (published September 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference. Thus, the term "Fc variant" comprises a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term "Fc variant" comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in disulfide bond formation, (2) incompatibility with a selected host cell N-terminal heterogeneity upon expression in a selected host cell, glycosylation, interaction with complement, binding to an Fc receptor other than a salvage receptor, or -19antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.

The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.

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

The term "dimer" as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in Figure 2.

The terms "derivatizing" and "derivative" or "derivatized" comprise processes and resulting compounds respectively in which the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; one or more peptidyl linkage is replaced by a non-peptidyl linkage; the Nterminus is replaced by -NRR, NRC(O)R', -NRC(O)OR, -NRS(0) 2 NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH-, wherein R and R 1 and the ring substituents are as defined hereinafter; the C-terminus is replaced by -C(O)R 2 or -NR 3

R'

wherein R 2

R

3 and R 4 are as defined hereinafter; and compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.

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

The term "randomized" as used to refer to peptide sequences refers to fully random sequences selected by phage display methods) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, RNApeptide screening, chemical screening, and the like.

The term "pharmacologically active" means that a substance so described is determined to have activity that affects a medical parameter blood pressure, blood cell count, cholesterol level) or disease state cancer, autoimmune disorders). Thus, pharmacologically active peptides comprise agonistic or mimetic and antagonistic peptides as defined below.

The terms "-mimetic peptide" and "-agonist peptide" refer to a peptide having biological activity comparable to a protein EPO, TPO, G-CSF) that interacts with a protein of interest. These terms further include peptides that indirectly mimic the activity of a protein of interest, such as by potentiating the effects of the natural ligand of the protein of interest; see, for example, the G-CSF-mimetic peptides listed in Tables 2 -21and 7. Thus, the term "EPO-mimetic peptide" comprises any peptides that can be identified or derived as described in Wrighton t al (1996), Sence 273: 458-63, Naranda et al. (1999), Proc. Natl. Acad. Sci. USA 96: 7569-74, or any other reference in Table 2 identified as having EPO-mimetic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

lariThe term "TPO-mimetic peptide" comprises peptides that can be identified or derived as described in Cwirla et (1997), Sciience 276:1696- 9, U.S. Pat. Nos. 5,869,451 and 5,932,946 and any other reference in Table 2 identifed as having TPO-mimetic subject matter, as well as the U.S. patent application, -Thrombopoietic Compounds," filed on even date herewith and hereby incorporated by reference. Those of ordinary skill in the art appreciate that each of these references enables'one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The term "G-CSF-mimetic peptide" comprises any peptides that can be identified or described in Paukovi et al. (1984), HoppeSeler

Z

PhBsi. Chem 365: 303-11 or any of the references in Table 2 identified as having G-CSF-mimetic subject matter. Those of ordinaryskil in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The tenrm CTLA4-mhimetic peptide" comprises any peptides that can be identified or derived as described in Fukumoto tal (1998), Niate Biotech. 16: 267-70. Those of ordinary skill in the art appreeiate-that each of these references enables one to select different peptides than actually -22disclosed therein by following the disclosed procedures with different peptide libraries.

The term "-antagonist peptide" or "inhibitor peptide" refers to a peptide that blocks or in some way interferes with the biological activity of the associated protein of interest, or has biological activity comparable to a known antagonist or inhibitor of the associated protein of interest. Thus, the term "TNF-antagonist peptide" comprises peptides that can be identified or derived as described in Takasaki et al. (1997), Nature Biotech.

15:1266-70 or any of the references in Table 2 identified as having TNFantagonistic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The terms "IL-1 antagonist" and "IL-lra-mimetic peptide" comprises peptides that inhibit or down-regulate activation of the IL-1 receptor by IL-1. IL-1 receptor activation results from formation of a complex among IL-1, IL-1 receptor, and IL-1 receptor accessory protein.

IL-1 antagonist or IL-lra-mimetic peptides bind to IL-1, IL-1 receptor, or IL-1 receptor accessory protein and obstruct complex formation among any two or three components of the complex. Exemplary IL-1 antagonist or IL-ra-mimetic peptides can be identified or derived as described in U.S. Pat. Nos. 5,608,035, 5,786,331, 5,880,096, or any of the references in Table 2 identified as having IL-lra-mimetic or IL-1 antagonistic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The term "VEGF-antagonist peptide" comprises peptides that can be identified or derived as described in Fairbrother (1998), Biochem. 37: -23- 17754-64, and in any of the references in Table 2 identified as having VEGF-antagonistic subject matter. Those of ordinary skill in the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

The term "MMP inhibitor peptide" comprises peptides that can be identified or derived as described in Koivunen (1999), Nature Biotech. 17: 768-74 and in any of the references in Table 2 identified as having

MMP

inhibitory subject matter. Those of ordinary skillin the art appreciate that each of these references enables one to select different peptides than actually disclosed therein by following the disclosed procedures with different peptide libraries.

Additionally, physiologically acceptable salts of the compounds of this invention are also encompassed herein. By "physiologically acceptable salts" is meant any salts that are known or later discovered to be pharmaceutically acceptable. Some specific examples are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate.

Structure of compounds In General. In the compositions of matter prepared in accordance with this invention, the peptide may be attached to the vehicle through the peptide's N-terminus or C-terminus. Thus, the vehide-peptide molecules of this invention may be described by the following formula

I:

I

2 )b wherein: F' is a vehicle (preferably an Fc domain); X' and

X

2 are each independently selected from

P

P

-P2, -(L1)cP(L')d 2-(L )e-P3, and

I

-24-

P

2 P3, and P 4 are each independently sequences of pharmacologically active peptides;

L

2

L

3 and L 4 are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1.

Thus, compound I comprises preferred compounds of the formulae

II

X

1

-F'

and multimers thereof wherein F' is an Fc domain and is attached at the Cterminus of X';

F'-X

2 and multimers thereof wherein F' is an Fc domain and is attached at the Nterminus of X 2

IV

F'-(L)c-P 1 and multimers thereof wherein F' is an Fc domain and is attached at the Nterminus of and

V

2 2 and multimers thereof wherein F' is an Fc domain and is attached at the Nterminus of -L-P-L-P 2 Peptides. Any number of peptides may be used in conjunction with the present invention. Of particular interest are peptides that mimic the activity of EPO, TPO, growth hormone, G-CSF, GM-CSF, IL-1ra, leptin, CTLA4, TRAIL, TGF-a, and TGF-P. Peptide antagonists are also of interest, particularly those antagonistic to the activity of TNF, leptin, any of the interleukins (IL-1, 2, 3, and proteins involved in complement activation C3b). Targeting peptides are also of interest, including tumor-homirig peptides, membrane-transporting peptides, and the like.

All of these classes of peptides may be discovered by methods described in the references cited in this specification and other references.

Phage display, in particular, is useful in generating peptides for use in the present invention. It has been stated that affinity selection from libraries of random peptides can be used to identify peptide ligands for any site of any gene product. Dedman et al. (1993), 1. Biol. Chem. 268: 23025-30. Phage display is particularly well suited for identifying peptides that bind to such proteins of interest as cell surface receptors or any proteins having linear epitopes. Wilson et al. (1998), Can. J. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins are extensively reviewed in Herz et al. (1997), 1. Receptor Signal Transduction Res. 17(5): 671-776, which is hereby incorporated by reference. Such proteins of interest are preferred for use in this invention.

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

-26- Table 3-Cytokine Receptors Classified by Receptor Code Cytokines (Iugands) Receptor Type family subfamrilly family subfamily I. Hematopoletic, 1. IL-2, IL-4, IL-7, I. Cytokine R 1 shared 7Cr cytokines IL-9, IL-i 3, IL- (OKR) 2. I.L-3, IL-5, GM- 2. shared GP 140 CSIF pR 3. IL-6, IL-li IL- 3. 3.shared RP 12, LIF, OSM, 130 ONTE, leptin (013) 4. G-CSF, EPO,. 4. "single chain" R TPO, PRL, GH IL-17, HVS-IL- 5. other Rc 17__ I I. I L-10 ligands IL-lO0, BORF-1, II.' IL-10 R Ill. Interferons 1. IFN4-al, d2, az4, Ill. Interferon R 1 IFNAF( 2. IFN-y 2. IFNGR IV. IL-i ligands 1 IL-ia, IL-10, JL- IV. IL-i R 1 Ra V. TNF ligands 1. TNF-a, TNF-3 V. NGF/TNF R' (ILT), FASI,

L,

CD3OL, CD27 VI. Chemokines 1. ax chemnokines: VI. Chemokine R 1. OXOR IL-8, GRO PF-4, SDF-1 2.p chemokines: 2. OCR MOP-i ,2,3,4,

RANTES,

eotaxin 3. y chemokines: 3. CR lymphotactin4.DR cIL-17R belongs to the CKR family but is not assigned to any of the 4 indicated subjamilies.

d Other IFN type I subtypes remain unassigned. Hematopoietic cytokines, IL-11 ligands and interferons do not possess functional intrinsic protein kinases. The signaling molecules for the cytokines are JAK's, STATs and related non-receptor molecules. IL-14, IL-16 and IL-18 have been cloned but according to the receptor code they remain unassigned.

'TNF-receptors use multiple, distinct intracellular molecules for signal transduction including "death domain" of FAS R and 55 kDa TNF-aR that participates in their cytotoxic effects. NGF/TNF R can bind both NGF and related factors as well as TNF ligands. Chemokine receptors are G protein-couplek, seven transmembrane (7TM, serpentine) domain receptors.

The Duffy blood group antigen (DARO) is an erythrocyte receptor that can bind several different chemokines. It belongs to the immunoglobulin superfamily but characteristics of its signal transduction events remain unclear.

-27- VII. Growth factors VII. RKF 1. TK sub-family 1.1 SCF, M-CSF, 1.1 IgTKIII R PDGF-AA AB, BB, FLT-3L, VEGF, SSV-

PDGF

1.2 FGFa, FGF 1.2 IgTK IV R 1.3 EGF, TGF-a, 1.3 Cysteine-rich VV-F19 (EGF-

TK-I

like) 1.4 IGF-I, IGF-II, 1.4 Cysteine rich Insulin

TK-II

NGF, BDNF, 1.5 Cysteine knot NT-3, NT-4 0 TK V 2. TGF-'1,p2,P3 2. STK subfamily" Exemplary peptides for this invention appear in Tables 4 through below. These peptides may be prepared by methods disclosed in the art. Single letter amino acid abbreviations are used. The X in these sequences (and throughout this specification, unless specified otherwise in a particular instance) means that any of the 20 naturally occurring amino acid residues may be present. Any of these peptides may be linked in tandem sequentially), with or without linkers, and a few tandemlinked examples are provided in the table. Linkers are listed as and may be any of the linkers described herein. Tandem repeats and linkers are shown separated by dashes for clarity. Any peptide containing a cysteinyl residue may be cross-linked with another Cys-containing peptide, either or both of which may be linked to a vehicle. A few crosslinked examples are provided in the table. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well; see, for example, EPO-mimetic peptides in Table 5. A few examples of intrapeptide disulfide-bonded peptides are specified in the table. Any of these peptides may be derivatized as described herein, and a few derivatized examples are provided in the table. Derivatizea peptides in The neurotrophic cytokines can associate with NGF/TNF receptors also.

-28the tables are exemplary rather than limiting, as the associated underivatized peptides may be employed in this invention, as well. For derivatives in which the carboxyl terminus may be capped with an amino group, the capping amino group is shown as For derivatives in which amino acid residues are substituted by moieties other than amino acid residues, the substitutions are denoted by a, which signifies any of the moieties described in Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9 and Cuthbertson et al. (1997), T. Med. Chem. 40:2876-82, which are incorporated by reference. The J substituent and the Z substituents Z, are as defined in U.S. Pat. Nos. 5,608,035,5,786,331, and 5,880,096, which are incorporated by reference. For the EPO-mimetic sequences (Table the substituents X 2 through X, and the integer are as defined in WO 96/40772, which is incorporated by reference. The substituents and are as defined in Sparks et al. (1996), Proc. Natl. Acad. Sci. 93: 1540-4, which is hereby incorporated by reference. X, X, X 6 and X, are as defined in U.S. Pat. No. 5,773,569, which is hereby incorporated by reference, except that: for integrin-binding peptides, X 1 X, X, X, X, X, X, and X, are as defined in International applications WO 95/14714, published June 1, 1995 and WO 97/08203, published March 6, 1997, which are also incorporated by reference; and for VIP-mimetic peptides, XI', XI", X, XX, and Z and the integers m and n are as defined in WO 97/40070, published October 30, 1997, which is also incorporated by reference. Xaa and Yaa below are as defined in WO 98/09985, published March 12, 1998, which is incorporated by reference. AA, AA,, ABI AB and AC are as defined in International application WO 98/53842, published December 3, 1998, which is incorporated by reference. X 2

X

3 and X 4 in Table 17 only are as- defined in European application EP 0 911 STKS may encompass many other TGF-p-related factors that remain unassigned. The protein kinases are intrinsic part of the intracellular domain of receptor kinase family (RKF). The enzymes participate in the signals transmission via the receptors.

-29- 393, published April 28,1999. Residues appearing in boldface are D amino acids.-All peptides are linked through peptide bonds unless otherwise noted. Abbreviations are listed at the end of this specification. In the "SEQ ID NO." column, "NR" means that no sequence listing is required for the given sequence.

Table 4-IL-1 antagonist peptide seq Iuences Sequence/structure

SEQ

ID NO: XXQZ YZ, XX 907 Z X.QZ YZA XX9098 TNFEWTPYJYWPAL 223 FAWTPGCYWQJYALIG 1 EWAYPGYWQJSYWPAL21 FEVGYNWQJYWPA 226 FEWTPGYWQJY 227 AFEWTPGYWQY 228 FEWTPaWYQJY21 FEWTPGWDYQJY23 AcFEWTPGWYQJY 241 FEWTPGW-pY-QJY 242 -FAWTPGYWQJY 243 FEWAPGYWQJY 244 FEWVPGYWQJY 245 FEWTPGYWQJY 246 AcFEWTPGYWQ)JY24 FEWTPAWYQJY 248 FEWTPSarW4YQJY 249 FEWTPGYYQPY 250 FEWTPGWWQPY 251 FEWTPNYWQPY 252 FEWTPVYWQJY 253 FEWTPecGYWQJY 254 FEWTPAibYWQJY 255 FEWTSarGYWQJY 256 FEWTPGYWQPYALPL 257 1 NapE WTPGYYQJY 258 .YEW4TPGYYOJY 259 FEWVPGYYQJY 260 FEWTPSYYQJY 261 .FEWTPNYYQJY 262 TKPR- 263 RKSSK 264 RKQDK 265 NRKQDK 266 RKQDKR 267 ENRKQDKRF 268 VTKFYF 269 VTKFY .270 VTDFY 271 SHLYWQPYSVQ 671 TLVYWQPYSLQT 672 RGDYWQPYSVQS67 VHVYWQPYSVQT 674 RLVYWQPYSVQT 675 SRVWFQPYSLOS67 NMVYWQPYSIQT 677 SVVFWQPYSVQT67 TFVYWQPYALPL 679 TLVYWQPYSIQR 680 RLVYWQPYSVQR 681 SPVFWQPYSIQI 682 WIEWWOPYSVQS 683 SLIYWQPYSLQM ~8 TRLYWQPYSVQR 685___ RCDYWQPYSVQT 686 MRVFWQPYS VON 687 KIVYWQPYSVQT 688 RHLYWQPYSVQR 689 -31- ARWYQPYSVQR 9 TRVWFQPYSVQ, 7102 GWQPYSVQE 711 GVWFOPYS VQR71 LRYWQPYSVQR 713 AVWWPYSQM 9 EYWO PYALPL69 SRIWWQ

PYALPL

735 SRQVQ PYALPL.

77 IARSWWQPYALPL 736 RGVYWQ TRLLWQPYALPL 738 RWFQ

PYALPR

-32- DAYWVQ PYALPL 740 WSGYFQ PYALPL 741 NIEFWQ PYALPL 742 TRDWVQ PYALPL 743 DSSWYQ PYALPL 744 IGNWYQ PYALPL 745 NLRWDQ PYALPL 746 LPEFWQ PYALPL 747 DSYWWQ PYALPL 748 RSQYYQ PYALPL 749 ARFWLQ PYALPL 750 NSYFWQ PYALPL 751 RFMYWQPYSVQR 752 AHLFWQPYSVQR 753 .WWQPYALPL 754 YYOPYALPL 755 YFQPYALGL 756 YWYQPYALPL -757 RWWQPYATPL 758 GWYQPYALGF 759 YWYQPYALGL 760 IWYQPYAMPL, 761 SNMQPYORLS -762 TFVYWQPY AVGLPAAETACN 763 TFVYWQPY SVQMTITGKVTM 764 TFVYWQPY SSHXXVPXGFPL 765 TFVYWQPY YGNPQWAIHVRH 766 TFVYWQPY VLLELPEGAVRA76 TFVYWQPY VDYVWPIPIAQV 768 GWYQPYVDGWR76 RWEQPYVKDGWS 770 EWYQPYALG WAR 771 GWWQPYARGL 772 LFEQPYAKALGL 773 GWEQPYARGLAG 77 AWVQPYATPLDE, 775 MWYQPYSSQPAE 776 GWTOPYSQQGEV77 DWFQPYSIQSDE 778 PWIQPYARGFG 779 RPLYWOPYSVQV 780 ThIYWQPSQ .781 RFDYWQPYSDQT 782 WHQFVQPYALPL 783 EWDS VWQPYSVQ TLLR 784 WEON VYWQPYSVQ SFAD 785 SDV VYWOPYSVQ SLEM 786 YYDG VYWQPYSVQ VMPA 787 SDIWYQ PYALPL 788 QRIWWQ PYALPL 789 -33- -34- GVTFSQ PYALPL 840 SIVWSQ PYALPL 841 SRDLVQ PYALPL 842 HWGH VYWQPYSVQ DDLG 843 SWHS VYWQPYSVQ SVPE 844 WRDS VYWQPYSVQ PESA 845 TWDA VYWQPYSVQ KWLD 846 TPPW VYWQPYSVQ SLDP 847 YWSS VYWQPYSVQ SVHS 848 YWYQOPY ALGL 849 YWY QPY ALPL 850 EWI OPY ATGL 851 NWE QPY AKPL 852 AFY QPY ALPL 853 FLY QPY ALPL 854 VCK OPY LEWC 855 ETPFTWEESNAYWQPYALPL 856 QGWLTWQDSVDMYWQPYALPL 857 FSEAGYTWPENTYWQPYALPL 858 TESPGGLDWAKIYWQPYALPL 859 DGYDRWRQSGERYWOPYALPL86 TAN VSSFE WTPGYWQPYALPL86 SVGEDHNFWTSE YWQPYALPL 862 MNDQTSEVSTFP YWQPYALPL 863 SWSEAFEQPRNL YWQPYALPL 864 QYAEPSALNDWG YWQPYALPL 865 NGDWATADWSNY YWQPYALPL 866 THDEHI YWQPYALPL 867 MLEKTYTTWTPG YWQPYALPL 868 WSDPLTRDADL YWQPYALPL 869 SDAF1TDSQAM YWQPYALPL 870 GDDAAWRTDSLT YWQPYALPL 871 AIIRQLYRWSEM YWQPYALPL 872 ENTYSPNWADSM YWOPYALPL 873 MNDQTSEVSTFP YWQPYALPL 874 SVGEDHNFWTSE YWQPYALPL87 QTPFTWEESNAY YWQPYALPL .876 ENPFTWQESNAY YWQPYALPL 877 VTPFTWEDSNVF YWQPYALPL 878 QIPFTWEQSNAY YWQPYALPL 879 QAPLTWQESAAY YWQPYALPL 880 EPTFTWEESKAT YWQPYALPL 881 TTTLTWEESNAY YWQPYALPL 882 ESPLTWEESSAL YWQPYALPL 883 ETPLTWEESNAY YWQPYALPL 884 EATFTWAESNAY YWQPYALPL 885 EALFTWKESTAY YWQPYALPL 886 STP-TWEESNAY YWQPYALPL 887 ETPFTWEESNAY YWQPYALPL 888 KAPF1WEESQAY YWQPYALPL 889 STSF1'VEESNAY YWQPYALPL 890 DSTF1'VEESNAY YWQPYALPL 891 -YIPFTWEESNAY YWQPYALPL 892 QTAFTWEESNAY YWQPYALPL 893 ETLFTWEESNAT YWQPYALPL 894 VSSFTWEESNAY YWQPYALPL 895 QPYA896 Py-1-NapPYQJYALPL 897 TAN VSSFE WTPG yWQPYALPL 898 FEWTPYWQPALPL899 FEWTPYWQJALPL900 -FEWTGYYQJALPL901 ETPFTWEESNAYYWQPYALPL 902 FTWEESNAYYWQJYALPL 903 ADVL YWQPYA GDVAE YWQPYA LPLTSL 905 SWTDYG YWQPYA LPISGL 906 FEWTPGYWQPYALPL 911 FEWTPGYWQJYALPL 912 FEWTPGWYQPYALPL 913 FEWTPGWYQJYALPL .914 FEWPGYYQPYALPL 915 FEWTPGYYQJYALPL 916 TAN VSSFEWTPGYWQPYALPL 918 SWTDYGYWQPYALPISGL 919 ETPFTWEESNAYYWVQPYALPL 920 ENTYSPNWADSMYWOPYALPL 921 SVGEDHNFWTSEYWQPYALPL 923 DGYDRWRQSGERYWQPYALPL 924 FE~v7GYWQPALPL925 FEWTPGYWQJY 926 FEWTPGYWQY 927 FEWTPGWYQJY 928 AEWTPGYWQJY .929 FAWTPGYWQJY 930 FEATPGYWQJY 931 FEWAPGYWQJY 932 FEWTAGYWQJY 933 FEWTPAYWQJY 934 FEWTPGAWOJY 935 FEWTPGYAQJY .936 FEWTPGYWQJA 937 FEWTGGYWVQJY 938 FEWTPGYWQJY 939 FEWTJGYWQJY 940 FEWTPecGYWQJY 941 FEWTPAibYWQJY 942 FEWTPSarWYQJYT 943 FEWTSarGYWQJY 944 -36- FEWTPNYWQJY 945 FEWTPVYWQJY 946 FEWTVPYWQJY 947 AcFEWTPGWYQJY 948 AcFEWTPGYWQJY 949 INap-EWTPGYYQJY 950 YEWTPGYYQJY 951 FEWVPGYYQJY 952 FEWTPGYYQJY 953 FEWTPsYYQJY 954 FEWTPrYYQJY 955 SHLY-Nap-rQPYSVQM 956 TLVY-Nap-QPYSLQT 957 RGDY-Nap-QPYSVQS. 958 VYWQPYSVQ 960 VY-Nap7QPYSVQ 961 TFVYWQJYALPL 962 FEVTPGYYQJ-Bpa 963 XaaFEWTPGYYQJ-Bpa 964 FEWTPGY-Bpa:.QJY 965 AcFEWTPGY-Bpa-QJY 966 FEWTPG-Bpa-!YQJY 967 ACFEWTPG-Bpa-YdjY- 968 AcFE-Bpa-TPGYYQJY, 969 AcFE-Bpa-TPGYYQJY 970 Bpa-EVWTPGYYQJY 971 AcBpa-EWTPGYYQJY 972 VYWQPYSVQ 973 RLVYWQPYSVQR 974 RLVY-Nap-QPYSVQR 975 RLDYWQPYSVQR 976 RLVWFQPYSVQR97 RLVYWQPYSIOR 978 DNSSWYDSFLL 980 DNTAWYESFLA 981 DNTAWYENFLL, 982 PARE DNTAWYDSFLI WC 983 TSEY DNTTWYEKFLA SO 984 SQIP DNTAWYQSFLL HG 985 SPEI DNTAWYENFLL TY 986 EQIY DNTAWYDHFLL SY 987 TPFI DNTAWYENFLL TY 988 TYTY DNTAWYERFLM SY 989 TMWO DNTAWYENFLL SY. 9 TI DNTAWYANLVQ TYPO 99-1 TI DNTAWYERFLA QYPD92 HI DNTAWYENFLL TYTP 993 SQ DNTAWYENFLL SYKA 994 01 DNTAWYERFLL QYNA 995 -37- NQ DNTAWYESFLL QYNT 996 TI DNTAWYENFLL NHNL 997 HY DNTAWYERFLQ QGWH 998 ETPFTWEESNAYYWQPYALPL 999 YI PFTWEESNAYYWQPYALPL 1000 DGYDRWRQSGERYWQPYALPL pY-INap-pY-QJYALPL 1002 TAN VSSFE WTPGYWQPYALPL 1003 FEWTPGYWQJYALPL 1004 FEWTPGYWQPYALPLSD 1005 FEWTPGYYQJYALPL 1006 FEWTPGYWQJY 1007 AcFEWVTPGYWQJY 1008 AcFEWTPGWVYQJY 1009 AcFEWVTPGYYQJY 1010 AcFEWTPaYWQJY 1011 AcFEWTPaWVYQJY 1012 AcFEWTPaYYQJY 1013 FEWTPGYYQJYALPL 1014 FEWTPGYWQJYALPL 1015 FEWTPGWYQJYALPL .1016 TAN VSSFE WTPGYWQPYALPL 1017 AeFEWTPGYWQJY. 1018 AcFEWTfPGWYQJY 1019 AcFEWTfPGYYQJY- 1020 AcFEWVTPAYWQJY 1021 AcFEWTPAWYQJY 1022 AcFEWTPAYYQJY 1023 -38- Table 5-EPO-mimetic peptide sequences Sequence/structure

SEQ

ID NO: YXCXXGPXTWXCXP 83 YXCXXGPX1VWXCXP-YXCXXGPXTh1XCXP 84 YXCXXGPXTWXCXP-A-YXCXXGPXTWXCXP YXC.XXG IPXTWXCXP-A;,-,, e 86

K

P3A 86 .YXCX XGPXTWXCXP-A-/ (a-amnine) .GGTYSCHFGPLTWVCKPOGG 87 GGDYHCRMGPLTWVCKPLGG 88 GGVYACRMGPITWVCSPLGG -m89 VGNYMCHFGPITWVCRPGGG GGLYLCRFGPVTWDCGYKGG 91 GGTYSCHFGPLTWVCKPQGG- 92

GGTYSCHFGPLTWVCKPQGG

GGTYSCHFGPL1WVCKPOGG 93 GGTYSCH FGPL1WVCKPQGG GGTYSCHFGPLTWVCKPQGGSSK 94 GGTYSCHFGPLTWVCKPQGGSSK-

GGTYSCHFGPLTWVCKPQGGSSK

GGTYSCHFGPLTWVCKPQGGSSK-A- 96

GGTYSCHFGPLTWVCKPQGGSSK

GGTYSCHFGPLTWVCKPQGGSS (samine) 9

K

PJA 97 GGTYSCHFGPLTWVCKPQ)GGSS/ (a-amine) GGTYSCHFGPLTWVCKPQGGSSK(-A-biotil) 98 CXXgGPXTWX,C 421 GGTYSCHGPL1VJVCKPOGG 422 VGNYMAHMGPITWVCRPGG 423 GGPHHVYACRMGPLTWIC 424 GGTYSCHFGPLTWVCKPQ 425 GGLYACHMGPMTWVCQPLRG 426 TIAQYICYMGPETWECRPSPKA 427 YSCHFGPLTWVCK 428 YCH FGPLTWVC 429 XX4X.,GPX,,WX,XP 124 YX,XXXG PXTWX,, 461 -39- XYxxX.

XGPXJWXXRXXA

XYX,CX X GPX TWX-rX-X.-XI

GGLYLCRFGPVTWDCGYKGG

GGTYSCHFGPLTWVCKPQGG

GGDYHCRMGPLTWVCKPLGGi GGVYACRMGPITWVCSPLGGi

VGNYMAHMGPITWVCRPGG~

GGTYSCHFGPLTWVCKPQ

GGLYACHMGPMTWVCQPLRG

[TIAQYICYMGPETWFECRPSPKA

YSCHFGPLTWVCK

YCHFGPLTWAVC

SCHFGPLTWVCK

(AX,),X.,XXGPXTWYXXA

419 4 1024 1025 1026 1029 1030 1035 1036 1037 1038 1039 1040 1041 1042 Table 6-TPO-mimetic peptide sequences Sequence/structure

SEQ

ID NO: IEGPTLRQWLAARA 13 IEGPTLRQWLAAKA 24 IEGPTLREWLAARA IEGPTLRQWLAARA-A-IEGPTLRQWLAARA 26 IEGPTLRQWLAAKA-A-IEGPTLRQWLAAKA 27 IEGPTLRQCLAARA-A-IEGPTLRQCLAARA 28 IEGPTLRQWLAARA-A-K(BrAc)-A-IEGPTLRQWLAARA 29 IEGPTLRQWLAARA-A-K( PEG) -A-IEGPTLRQWLAARA IEGPTLRQCLAARA-A-IEGPTLRQ)WLAARA 31 IEGPTLRQC;LAAR'A-A-IEGPTLRQ)WLAARA 31 IEGPTLRQWLAARA-A-I EGPTLRQCL AARA 32 IEGPTLRQWLAARA-A-iEGtuILRQCLAARA 32 VRDQIXXXL 33 TLREWL 34 GRVRDQVAGW GRVKDQIAQL 36 GVRDQVSWAL 37 ESVREQVMKY 38 SVRSQISASL 39 GVRETVYRHM GVREVIVMHML 41 GRVRDQIWAAL 42 AGVRDQILIWL 43 GRVRDQIMLSL 44 GRVRDOI(X),L CTLRQWLQGC 46 CTLQEFLEGC 47 CTRTEWLHGC 48 CTLREWLHGGFC 49 CTLREWVFAGLC CTLRQWLILLGMC 51 CTLAEFLASGVEQC 52 CSLQEFLSHGGYVC 53 CTLREFLDPTrAVC 54 CTLKEWLVSHEVWC CTLREWL(XX,,C 56-60 REQ PTLRQWM 6 EGPTLRQWLA 62 ERGPFWAKAC 63 REGPRCVMWM 64 CGTEGPTLSTWLDC -41- CEQDG PTLLEWLKC

-CELVGPSLMSWLTCL

CLTGPFVTQWLYECU

CRAGPTLLEWLTLC;

CADGPTLREWISFC

C(X)

1 -,EGPTLREWL(X) 12

C

GGCTLREWLHGGFCGG

GGCADGPTLREWISFCGG

GNADGPTLRQWLEGCRRPKN

LAIEGPTLRQWLHGNGRDT

HGRVGPTLREWKTQVATKK

TIKGPThRQWLKSREHTS.

ISDGPTLKEWLSVTIRGAS

SIEGPTLREWLTSRiTPHS 66 67 68 69 76 77 78 79 81 82 -42- Table 7-G-CSF-mimetic peptide sequences Sequence/structure

SEQ

ID NO: EEDCK 99 EEDCK 99 EEDCK 99 EEDaK 100 EEDcTK 100 EEDaK 100 pGIuEDaK 101 pGIuEDaK 101 PGIuEDaK 101 PicSDaK 102 PicSDcrK 102 EEDCK-A-EEDCK 103 EEDXK-A-EEDXK 104 -43- Table 8-TNF-an tagonist peptide sequences Sequeflce/stfflctlre

YCFTASENHCY

YCFTNSENHCY

YCFTRSENHCY

FCASENHCY

YCASENHCY

FCNSENHCY

FCNSENRCY

FCNSVENRCY

yCSQSVSNDCF

FCVSNDRCY

IYCrarEL

GQVC~Y

-44- Table 9--Integrin-binding peptide sequences Sequence/structure SEQ ID NO: RXIETX,WX, 441 RXETXWX, 442 RGDGX43 CRGDGXC 4.44 CXXRLDX XC 445 CARRLDAPC 446 CPSRLDSPC 447

X

1 XXRGDXX,X, 448 CX,CRGDCXC 449 CDCRGDCFC 450 CDCRGDCLC 451 CLCRGDCIC 452 XXDX4X,,,X,453 XIX)X DDXX.,X,),X, 454 CWDDGWLC 455 CWDDLWWLC -456 CWDDGLMC 457 CWDDGWMC 458 CSWDDGWLC 459 CPDDLWWLC 460 NGR

NR

GSL

NR

RG D

NR

CGRECPRLCQSSC 1071 CNGRCVSGCAGRC 1072 CLSGSLSC 1073 RGD

NR

NGR

NR

GSL

NR

NGRAHA 1074 CNGRC 1075 ODORGOOFO 1076 CGSLVRC 17 DLXXL 1043 RTDLDSLRTYTL 1044 RTDLDSLRTY RTDLDSLRT 1054 RTDLDSLR 1078 GDLDLLKLRLTL 1079 GDLHSLRQLLSR 1080 RDDLHMLRLQLW 18 SSDLHALKKRYG 1082 RGDLKQLSELTW 1083 RGDLAALSAPPV 1084 Table lO-Selectin antagonist peptide sequences Sequence/structure .SEQ ID NO: DITWDQLWDLMK 147 DITWDELWKIMN 148 DYTWFELWDMMQ 149 QITWAQLWNMMK 150 DM1'WHDLWTLMS 151 DYSWHDLWEMMS 152 EITWDQLWEVMN HVSWEQLWDIMN 154 HITWDQLWRIMT 155 RNMSWLELWEHMK 156 AEWTWDQLWHVMNPAESQ 157 HRAEWLALWEQMSP 158 KKEDWLALWRIMSV 159 ITWDQLWDLMK 1.60 D11'WDQLWDLMK 161 DITWDQLWDLMK 162 DITWDQLWDLMK 163 CQNRYTDLVAIQN.KNE 462.

AENWADN EPNNKRNNED.46 RKNNKTWTWVGTKKALTNE .464 KKALTN EAEN WAD 465 CQXRYTDLVAIQNKXE 466 RKXNXXWTWVGTXKXLTEE 467 AENWADGEPNNKXNXED 468 CXXXYTXLVAIQNKXE 469 RKXXXXWXWVGTXKXLTXE .470 AXNWXXXEPNNXXXED 471 XKXKTXEAXNWXX 472 -46- Table li-Antipathogenic peptide sequences S -equen ce/structure SEQ ID NO: GFFALIPKI ISSPLFKTLLSAVGSALSSSGGQQ 503 GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE 504 GEFALI PKIISSPLFKTLLSAV 505 GFFALIPKI ISSPLFKTLLSAV 506 KOFFALIPKIISSPLFKTLLSAV 507.

KKGFFALIPKIISSPLFKTLLSAV 508 KKGFFALIPKIISSPLFKTLLSAV 509.

GFFALIPKIIS' 510 GIGAVLKVLTTGLPALISWIKRKRQQ51 GIGAVLKVLTTGLPALISWIKRKRQQ 512 GIGAVLKVLTTGLPALISWIKRKRQQ 513 GIGAVLKVLTTGLPALISWIKR 514 AVLKVLTTGLPAUSWIKR 515 KLLLLLKLLLLK' 516 KLLLKLLLKLLK .517 KLLLKLKLKLLK 518 KKLLKLKLKLKK 519 KLLLKLLLKLLK 520, KLLLKLKLKLLK 521; KLLLLKI 522, KLLLKLLK 523' KLLLKLKLKLLK 524 KLLLKLKLKLLK 525: KLLLKLKLKLLK 526 KAAAKAAAKAAK 527 KVVVKVWVKVVK 528 KVVVKVKVKVVK 529 KVVVKVKVKVK 5380 KVVVKVKVKVVK 531 KLILKL-, 532 KVLHLL, 533 LKLRLL 534 KPLHLL., 535 KLILKLVR 536 KVFHLLHL. 537 H KFRILKL 538 KPFHILHL53 KIIIKIKIKIIK 540 KIIIKIKIKIIK 541 KIIIKIKIKIIK 542 *KIPIKIKIKIPK 543 KIPIKIKIKIVK 544___ RIIIRIRIRIIR 545 RIIIRIRIRIIR, 546 RIIIRIRIRIIR 547 RIVIRIRIRLIR 548 -47- RIIVRIRLRIIR 549 RIGIRLRVRIIR 550 -KIVIRIRIRLIR 551 RIAVKWRLRFIK 552 KIGWKLRVRIIR 553 KKIGWLIIRVRR 554 RIVIRIRIRLIRIR 555 RIIVRIRLRIIRVR 556 RIGIRLRVRIIRRV 557 KIVIRIRARLIRIRIR 558 RIIVKIRLRIIKKIRL 559 KIGIKARVRIIRVKII 560 RIIVHIRLRIIHHIRL 561 HIGIKAHVRIIRVHII 562 RIYVKIHLRYIKKIRL 563 KIGHKARVHIIRYKII 564 RIYVKPHPRYIKKIRL 565 KPGHKARPHIIRYKII 566 KIVIRlRIRLIRIRIRKIV 567 RIIVKIRLRIIKKIRLIKK 568 KIGWKLRVRIIRVKIGRLR 569 KLVIRIRIRLIRIRIRKIVKVKRIR 570 RFAVKIRLRIIKKIRLIKKIRKRVIK 571 KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK 572 RIYVKPHPRYIKKIRL 573 KPGHKARPHIIRYKII 574 KIVIRIRIRLIRIRIRKIV 575 RIIVKIRLRIIKKIRLIKK 576 RIYVSKISIYIKKIRL 577 KIVIFTRIRLTSIRIRSIV 578: KPIHKARPTIIRYKMI 57 cyclicCKGFFALIPKIISSPLFKTLLSAVC 580 CKKGFFALIPKIISSPLFKTLLSAVC 581 CKKKGFFALIPKIISSPLFKTLLSAVC 582 CyciIcCRIVIRIRIRLIRIRC 583 CyclicCKPGHKARPHI IRYKIIC 584 CyclicCRFAVKIRLRIIKKIRLIKKIRKRVIKC 585, KLLLKLLL KLLKC 586 KLLLKLLLKLLK 587 KLLLKLKLKLLKC 588 KLLLKLLLKLLK 589 -48- Table 12-VIP-mimetic peptide sequences Sequence/structure SEQ ID NO: HSDAVFYDNYTR LRKQMAVKKYLN'SILN 500 NMe HSDAVFYDNYTR LRKQMAVKKYLN SILN 591 X, 4, X, .592' X, S X, LN 593 NH OH CO KKYX5 NH OH CO X6 594 (CH2)m Z -(CH2)n KKYL 595 NSILN 596 KKYL 597 KKYA 598 AVKKYL599 NSILN .600 KKYV 601 SI LauN 602 KKYLNIe 603 NSYLN 604 NSIYN .605 KKYLPPNSILN 606 LauKKYL 607 CapKKYL 608 KYL NR KKYNIe 609 VKKYL 610.

LNSILN 611 YLNSILN 612 KKYLN 613 KKYLNS 614 KKYLNSI 615 KKYLNSIL 616 KKYL 617 KKYDA .618 AVKKYL 619 NSILN 620 KKYV 621 SILauN 622 NSYLN 623, NSIYN 624 KKYLNIe 625 KKYLPPNSILN 626 KKYL 627 KKYDA 628- AVKKYL 629 NSILN 630 KKYV 631 SILauN 632 -49- LauKKYL 633 CapKKYL 634 KYL -NR KYL

NR

KKYN1e 635 VKKYL 636 LNSILN. 637 YLNSILN 638 KKYLNIe 639 KKYLN640 KKYLNS641 KKYLNSI 642 KKYLNSIL 643 KKKYLD 644 cyclicCKKYLC 645 CKKYLK 646 KKYA 647 WWTDTGLW 648' WWTDDGLW 649 WWDTRGLWVWTI 65,0 FWGNDGIWLESG 651 DWDQFGLWRGAA 652 RWDDNGLWVVVL 653___ GGRWDQGLWVA655 KLWSEQIWMGE656 CWSMHLWLC657 GCWDNTIWVPC658 DWDTRLWVY659 SLWDENGAWI 660 KWDDRGLWMH 661 QAWNERGLWT 662 QWDTRGLWVA 663 WNVHGIWQE, 664 SWDTRGLWVE 665 DWDTRGLWVA 666 SWGRDGLWIE 667 EWTDNGLWAL 668 SWDEKGLWSA 669 SWDSSGLWMD 1670H Table 13-Mdmfhdm antagonist peptide sequences Sequence/structure SEQ ID NO: TFSDLW 130 QETFSDLWKLLP 131____ QPTFSDLWKLLP 132 QETFSDYWKLLP ~133, QPTFSDYWKLLP 134 M PRFMDYWEGLN 135 VQNFI DYWTQQF 136 TGPAFTHYWATF 137 IDRAPTFRDHWFALV 138 PRPAL VFADY WETLY 139 PAFSRFWSDLSAGAH 140 PAFSRFWSKLSAGAH 141 PXFXDYWXXL 142 QETFSDLWKLLP 143 OPTFSDLWKLLP 144 QETFSDYWKLLP 145- QPTFSDYWKLLP 146H Table 14-Calmodulin antagonist peptide sequences Sequence/structure SEQ

IVDNO:

SCVKWG KKEFCGS .164 SCWKYWGKECGS 165 SCYEWGKLRWCGS 166 SCLRWGKWSNCGS 167 SOWRWGKYQICGS 168 SC VS WGALKLCGS 169 SCIRWGQNTFCGS 170.

SCWQWGNLKICGS 171 SCVRWGQLSICGS 172 LKKFNARRKLKGAILTTMLAK 173 RRWKKNFIAVSAANRFKK .174 RKWQKTGHAVRAIGRLSS 175 INLKALAALAKKIL 176 KIWSILAPLG1TLVKLVA 177 LKKLLKLLKKLLKL 178 LKWKKLLKLLKKLLKKLL 179 AEWPSLTEIKTLSHFSV 180 .AEWPSPTRVISTTYFGS 181 AELAHWPPVKTVLRSfT 182- AEGSWLQLLNLMKQMNN 183 AEWPSLTEIK 14 -51- Tablfe 15-Mast cell antagonists/Mast cell protease inhibitor peptide sequences Sequence/structure SEQ ID NO: SGSGVLKRPLPILPVTR 272 RWLSSRPLPPLPLPPRT 273 GSGSYDTLALPSLPLHPMSS 274 GSGSYDTRALPSLPLHPMSS 275 GSGSSGVTMYPKLPPHWSMA 276 GSGSSGVRMYPKLPPHWSMA 277 GSGSSSMRMVPTI PGSAKHG 278 RNR

NR

QT

NR

ROK NR NRQ

NR

RQK

NR-

RNRQKT 436 RNRQ 437 RNRQK 438 NRQKT 439 RQKT 440 -52- -Table 16-SH3 antagonist peptide sequences Sequence/structure SEQ ID NO: RPLPPLP 282 RELPPLP 283 SPLPPLP 284.

GPLPPLP 285 RPLPIPP. 286 RPLPIPP 287 RRLPPTP 288 RQLPPTP 289 RPLPSRP 290 RPLPTRP 291 SRLPPLP 292 RALPSPP29 RRLPRTP 294' RPVPPIT 295 ILAPPVP 296, RPLPMLP 297 RPLPILP .298 RPLPSLP .299 RPLPSLP 300' RPLPMIP 301 RPLPLIP: 302 RPLPPTP 303 RSLPPLP 304 RPQPPPP 305 RQLPIPP 306 XXXRPLPPLPXP 307 XXXRPLPPIPXX 308 XXXRPLPPLPXX 309 RXXRPLPPLPXP 310 RXXRPLPPLPPP 311 PPPYPPPPIPXX 312 PPPYPPPPVPXX 313 LXXRPLPX[P 314 'IXXRPLPXLP 315 PPX9XP PP'P 316 +PP'PPXKPXWL 317 RPXTLPT~R+SXP 318 PPVPPRPXXTL 319 '1P'LP'VK 320 +eDXP LPXLP 321 -53- Table 17-Somatostatifl or cortistatin mimetic peptide sequences Sequence/structure

SEQ

ID NO:

X'-X

2 -L sT rPh- 3 S r-X 4 474 As Ar Mt~roC sr Asnph pr Ly sThrhereC sL s lei Met ProC se. ro y Asnnpher LPh sTr Iee er Sr s 475 Me~rC L ~sPhe Phe T Ly sThr PhereC 475 Met Pro Cy Arg Asn Lhe PheT' L _ir~e Ser rCy sLys C sArg Asn Phe Phe Tr LsThr Phe r Ser Cs47 As AU~ Met Pro C sL srlAsn Phe Phe Tr L s ehrSer C sL 9 Met Pro C sL s he PheTr L ~r Phe r Ser C SL 473 C s AsAsnPhe PheTr LsThr Phe Sr er rCSL47 Asp Ar Met ProCG s L s Asn Phe Phe T Lv sThr PheShr Ser Cy 48 Met Pro Cy s AsnPhe PheTr Ly sThr Phe rSer Cys 496 C s sAsn Phe Phe Tr Lvv shihe SerSer C s 497 -54- Table 18-UKR antagonist peptide sequences Sequence/structure SEQ ID NO:.

AEPMPHSLNFSQYLWYT 196 AEHTYSSLWDTYSPLAF 197 AELDLWMRHYPLSFSNR 198 AESSLWTRYAWPSMPSY 199 AEWHPGLSFGSYLWSKT 200 AEPALLNWSFFFNPGLH 201 AEWSFYNLHLPEPQTIF 202 AEPLDLWSLYSLPPLAM 203 AEPTLWQ)LYQFPLRLSG 204 AEISFSELMWLRSTPAF 205 AELSEADLWTTWFGMGS 206,.

AESSLWRI FSPSALMMS 207 AESLPTLTSILWGKESV 208 AETLFMDLWHDKHILLT 209 AEILNFPLWHEPLWSTE 210 AESQTGTLNTLFWNTLR 211.

AEPVYQYELDSYLRSYY- 430 AELDLSTFYDIQYLLRT 431, AEFFKLGPNGYVYLHSA 432 FKLXXXGYVYL. 433 AESTYHHLSLGYMYTLN 434 YHXLXXGYMYT 435 Table 19-Macrophage and/or T-cell inhibiting peptide sequences Sequence/structure

SEQ

ID NO: Xaa-Yaa-Arg

NR

Arg-Yaa-Xaa

NR

Xaa-Arg-Yaa

NR.

Yaa-Arg-Xaa

NR

Ala-Arg -NR Arg-Arf

NR

Asn-Arg-

NR

Asp-Arg...

NR

Cys-Arg

NR

GIn-Arg -NR.

Glu-Ar

NR

Gly-Arg

NR

His-arg

NR.

Ile-Akg

-NR

LeuwArg.

NE

Lys-ArgNR Met-At.

NE.

Phe-ArgNR Ser-ArgNR Thr-'ArgNR Trp-ArgNE Tyr-ArgNE Val-Ar'NR Ala-Glu-Arg

NR

Arg-Glu-Arg.

NE

Asn-Glu-Arg

N

Asp-Glu-Arg

NE

Cys-Glu-Arg

NE

Gln-Glu-Arg

NE

Glu-Glu-Arg

-NE

Gly-Glu-Arg

NE

His-Glu-r

NE

Ile-Glu-Arg

NE

Leu-GuArg

NE

Lys-Glu-Arg

NE

Met-Glu-Arg

NER

Phe-Glu-Arg

NE

Pro-Glu-Arg'

NE

Ser-Glu-Arg

-NE

Thr-Glu-Arg

NE%

Trp-Glu-Arg-

NE

Tyr-Glu-Arg

NE

VaI-Glu-Arg-

NE

-56- Arg-Ala NR Arg-Asp NR Arg-Cys NR Arg-Oln NR Arg-Glu NR Arg-G y NR Afd-Hiis NR Arg-lle .NR Arg-Leu, NR Arg-Lys NR Arg-met NR Aig-Phed NR Arg-Peo NR Arg4Ser NR Ard-Thr NR Arg-Tto. NR Ae§-Tyt NR At -Val MR Arg- Glu-Ala NR Ard-Glu-Asn NR Arg-Glu-Asp NR Arg-GIU-Cys NR At ,-010-Gln MR Ard-GOu-Glu NR -Arg-GIU-Gly MR Arg-Glu-His NR Arg-Glu-lle MR ArGlu-Leu MR Arg?-Gju-Lys MR Arg-Glu-Met MR Ard-Glu-Phe MR Arg-.GIU-Pro NR Arg-Glu-Ser MR Arg-Glu-Thr N~R Arg-Glu-Trp MR Arg-Glu-Tyr MR Arg-Glu-Val' MR AIA-Arg-Glu MR: Arg-Arg-Glu MR Asn-Arg-Glu MR As -Arg-Glu MR Cys-Arg-Glu MR Gin-Arg-Glu MR GlU-Arg-Glu MR Gly-Arg-Glu NR' His-Arg-Glu R Ile-Arg-Glu MR Leu-Arg-Glu NR Lys-Arg-Glu MR Met-Arg-Glu MR -57- 58 Table 20--Additional Exemplary Pharmacologically Active Peptides Sequence/structure SEQ Activity

ID'

NO:!

VEFNCDIHVMWEWECFERL VEGF-antagonist GERWCFDGPLTWVCGEES 398 1VEGF-antagonist RGWVEICVADDNGMCVTEAO '1085,1 VEGF-antagodist GWDEODVAR-MWEWECFAGV 1086 VEGF- antagonist GERWCFDGPRAWVCGWEI 501 VEGF- antagonist EELWCFDGPRAWVCGYVK 502 jVEGF- antagonist RGWVEICAADOYGRCLTEAQ 1031 VEGF-, antagonist RGWVEICESOVWGRCL 1087 VEGF- antagonist RGWVEICESOVWGRCL 1088 VEGF- antagonipt GGNECDIARMWEWECFERL 1089 VEGF- antagonist RGWVEICAADDYGRCL 1090 VEGF-antagonist CTTHWGFTLC 1028 MMP inhibitor CLRSGXGC 101 MMP inhibitor CXXHWGFXXC 1092 MMP inhibitor 1093' MMP inhibitor CRRWGFFC1094 MMP inihibitor 1095 MMP inhibitor CSLWGFWC1096 CTLA4-mimetc GFVCSGIFAVGVG 125 CTLA4-rnimetic APGVRLGCAVLGRYC 126 CTLA4-mimetic 105 Antvral (HBV) ICVVQDWGHHRCTAGHMANLTSHASAI 127 C3b antagonist ICVVQDWGHHRCT 128' C3b antagonist.

CWVQDWGHHAC 129 C3b antagonist STGGFODVYDWARGVSSAL1TTLVATR 185 Vinculin-binding STGGFDDVYDWARRVS8ALTTTLVATR 186 Vinculln-binding SRGVNFSEWLYDMSAAMKEASNVFPSRRSR 187 Vinculin-binding SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR 188 Vinculin-binding SSPSLYTQFLVNYESAATRIQDLLIASRPSR 189 Vinculin-binding SSTGWVDLLGALQRAADAThTSIPPSLQNSR 190 Vinculin-binding DVYTKKEUECARRVSEK 191 Vincuiln-binding EKGSYYPGSGIAQFHIDYNNVS 192 C4BP-binding SGIAQFHIDYNNVSSA:EGWHVN 193 C4131-binding LVTVEKGSYYPGSGIAQFHlDYNNVSSAEGWHVN 194 C413P-binding SGIAQFHIDYNNVS 195 C4B3P-bindlng LLGRMK 279 ant!-HBV ALLGRMKG 280 anti-HBV LDPAFR 281 anti-HBV CXXRGO.G 322 Inhibition of platelet aggregation RPLPPLP E323 Src antagns PPVPPR 324 Src antagonist XFXDXWXXLXX f35- Ant-cancer (particularly for -59sarcomas) KACRRLFGPVDSEQLSRDCD 326 p1 6-mimetic RERWNFDFVTETPLEGDFAW 327 p16-mimetic .KRRQTSMTDFYHSKRRLIFS 328 p1 6-mimetic TSMTDFYHSKRRLIFSKRKP 329 p1 6-mimetic RRILIF 330 p16-mimetic KRRQTSATDFYHSKRRLIFSRQIKiWFQNRRMKWKK 331 p1 6-mnimetic KRRLIFSKRQIKIWFQNRRMKWKK 332 p1 6-mimetic Asn Gin Gly Arg His Phe Cys Gly Gly Ala Leu Ilie His Ala 498 CAP37 mimeticlLPS Ara Phe Val Met Thr Ala.Ala Ser Cys Phe Gin, binding Arg His Phe Cys Gly Gly Ala Leu Ilie His Ala Arg Phe Val 499 CAP37 mimeticlLPS Met Thr Ala Ala Ser Cys. binding Gly Thr Arg Cys Gin Val Ala Gly Trp Gly Ser Gin Arg Ser 500 CAP37 mimeticfLPS Giy Gly Arg Leu Ser Arg Phe Pro Arg Phe Val Asn Val binding WHWRHRIPLQLAAGR 1097 carbohydrate (GID1 alpha) mimetic LKTPR3V 1098 D2GPI Ab binding NTLKTPRV 1099 f32GPI Ab binding NTLKTPRVGGC 1100 132GPI, Ab binding KDKATF 1101 I32 GPI Ab binding KDKATFGCHD 1102 I32GPI Ab binding KDKATFGCHDGC 1103 132GPI Ab binding TILRVYK .1104 132GPI Ab binding ATLRVYKGG 1105. I2GPI Ab binding CATLRVYKGG 1106 i32GPI Ab binding INLKALAALAKKIL 1107 Membranetransporting GWT NR Membranetransporting GWTLNSAGYLLG 1108 Membranetransporting GWTLNSAGYLLGKINLKALAALAKKIL 1109 Membranetransporting The present invention is also particularly useful with pep tides having activity in treatment of: cancer, wherein the peptide is a VEGF-mimeticor a VEGF receptor antagonist, a HER2 agonist or antagonist, a CD20 antagonist and the like; asthma, wherein the protein of interest is a CKR3 antagonist, an receptor antagonist, and the like; thrombosis, wherein the protein of interest is a GPIIb antagonist, a GPIIIa antagonist, and the like; autoimmune diseases and other conditions involving immune modulation, wherein the protein of interest is an IL-2 receptor antagonist, a CD40 agonist or antagonist, a CD40L agonist or antagonist, a thymopoietin mimetic and the like.

Vehicles. This invention requires the presence of at least one vehicle

(F

1

F

2 attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the amino acid residues. Multiple vehicles may also be used; Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or a sidechain. An Fc domain is the preferred vehicle. The Fc domain may be fused to the N or C termini of the peptides or at both the N and C termini. For the TPO-mimetic peptides, molecules having the Fc domain fused to the N terminus of the peptide portion of the molecule are more bioactive than other such fusions, so fusion to the N terminus is preferred.

As noted above, Fc variants are suitable vehicles within the scope of this invention. A native Fc may be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631 and WO 96/32478.

In such Fc variants, one may remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or Damino acids. Fc variants may be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which: 1. Sites involved in disulfide bond formation are removed. Such removal may avoid reaction with other cysteine-containing proteins present in -61the host cell used to produce the molecules of the invention. For this purpose,.the cysteine-containing segment at the N-terminus may be truncated or cysteine residues may be deleted or substituted with other amino acids alanyl, seryl). In particular, one may truncate the Nterminal 20-amino acid segment of SEQ ID NO: 2 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2.

Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.

2. A native Fc is modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the Nterminus of a typical native Fc, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One may also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 2 (Figure 4) is one such Fc variant.

3. A portion of the N-terminus of a native Fc is removed to prevent Nterminal heterogeneity when expressed in a selected host cell. For this purpose, one may delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 4. One or more glycosylation sites are removed. Residues that are typically glycosylated asparagine) may confer cytolytic response.

Such residues may be deleted or substituted with unglycosylated residues alanine).

Sites involved in interaction with complement, such as the Clq binding site, are removed. For example, one may delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and sor may be avoided with such an Fc variant.

-62- 6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc may have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so may be removed.

7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so may be removed.

8. When the native Fc is derived from a non-human antibody, the native Fc may be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.

Preferred Fc variants include the following. In SEQ ID NO: 2 (Figure 4) the leucine at position 15 may be substituted with glutamate; the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, one or more tyrosine residues can be replaced by phenyalanine residues.

An alternative vehicle would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued April 14, 1998 to Presta et aL Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of "vehicle" and are within the scope of this invention. Such vehicles should be selected for increased half-life by avoiding sequences recognized by proteases) and decreased immunogenicity by favoring nonimmunogenic sequences, as discovered in antibody humanization).

-63- As noted above, polymer vehicles may also be used for F' and F'.

Various means for attaching chemical moieties useful as vehicles are currently available, see, Patent Cooperation Treaty ("PCT") International Publication No. WO 96/11953, entitled "N-Terminally Chemically Modified Protein Compositions and Methods," herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water soluble polymers to the N-terminus of proteins.

A preferred polymer vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kDa to about kDa. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound an aldehyde, amino, or ester group).

A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis (see, for example, Figures 5 and 6 and the accompanying text herein). The peptides are "preactivated" with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by -64analytical HPLC, amino acid analysis and laser desorption mass spectrometry.

Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by al-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kD to about 70 kD. Dextran is a suitable water soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic inmmunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference. Dextran of about 1 kD to about 20 kD is preferred when dextran is used as a vehicle in accordance with the present invention.

Linkers. Any "linker" group is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. The linker is preferably made up of amino acids linked together by peptide bonds.

Thus, in preferred embodiments, the linker is made up of from 1 to amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In a more preferred embodiment, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. Even more preferably, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Thus, preferred linkers are polyglycines (particularly (Gly)S), poly(Gly-Ala), and polyalanines.

Other specific examples of linkers are: (Gly) 3 Lys(Gly), (SEQ ID NO: 333); (Gly),AsnGlySer(Gly), (SEQ ID NO: 334); (Gly)3 Cys(Gly), (SEQ ID NO: 335); and GlyProAsnGlyGly (SEQ ID NO: 336).

To explain the above nomenclature, for example, (Gly),Lys(Gly) means Gy-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are also preferred. The linkers shown here are exemplary; linkers within the scope of this invention may be much longer and may include other residues.

Non-peptide linkers are also possible. For example, alkyl linkers such as wheein s 2-20 could be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl lower acyl, halogen Cl, Br), CN,

NI-,

phenyl, etc. An exemplary non-peptide linker is a PEG linker,

SVI

N 0n

H

wherein n is such that the linker has a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may be altered to for derivatives in the same manner as described above.

Derivatives. The inventors also contemplate derivatizing te peptide and/or vehicle portion of the compounds. Such derivatives may improveptide and/solubility, absorption, biological half life, and the like of the compounds. The moieties may alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. Exemplary derivatives include compounds in which: 2 5 1. The compound or some portion thereof is cyclic. For example, the peptide portion may be modified to contain two or more Cys residues in the linker), which could cyclize by disulfide bond formation.

-66- For citations to references on preparation of cyclized derivatives, see Table 2.

2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion may be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound may also be cross-linked through its C-terminus, as in the molecule shown below.

VII

O

H 0

NH

2 L -yNH 3;: 4. One or more peptidyl linkages (bonds) is replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages are -CH,carbamate phosphonate, -CH,-sulfonamide urea -CH,-secondary amine, and alkylated peptide wherein R 6 is lower alkyl].

The N-terminus is derivatized. Typically, the N-terminus may be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include -NRR 1 (other than -NH 2

-NRC(O)R',

-NRC(O)OR, -NRS(O) 2

R

1 -NHC(O)NHR', succinimide, or benzyloxycarbonyl-NH- (CBZ-NH-), wherein R and R are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from the group consisting of C-C, alkyl, C 1 alkoxy, chloro, and bromo.

6. The free C-terminus is derivatized. Typically, the C-terininus is 2 5 esterified or amidated. For example, one may use methods described in the art to add (NH-CH 2

-CHI-NH)

2 to compounds of this invention -67having anyof SEQ ID NOS: 504 to 508 at the C-terminus. Likewise, one may use methods described in the art to add -NH 2 to compounds of this invention having any of SEQ ID NOS: 924 to 955, 963 to 972, 1005 to 1013, or 1018 to 1023 at the C-terminus. Exemplary C-terminal derivative groups include, for example,

-C(O)R

2 wherein

R

2 is lower alkoxy or -NR 3 R wherein R and R are independently hydrogen or Cj-

C

8 alkyl (preferably

C-C

4 alkyl).

7. A disulfide bond is replaced with another, preferably more stable, crosslinking moiety an alkylene). See, Bhatnagar et al.

(1996), 1. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am.

SmP, 357-9.

8. One or more individual amino acid residues is modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.

Lysinyl residues and amino terminal residues may be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-aminocontaining residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; Omethylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3butanedione, 1, 2 cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents of the high pKa of the guarginepsilon-amino may react with the groups of lysine as well as the argininePsilon-amio group.

-68- Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides such as 1-cyclohexyl- 3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia- 4 4 dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize crosslinking. See, Bhatnagar et al. (1996), J. Med. Chem. 39:3814-9.

Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular vehicles. Commonly used cross-linking agents include, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,

N-

hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- 2 5 dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-Nmaleimido-1,8-octane. Derivatizing agents such as methyl-3-[(pazidophenyl)dithio]propioimidate yield photoactivatable ntermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates -69and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins.

Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X- Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and 0linked oligosaccharides and, by virtue of its negative charge, may confer acidic properties to the glycosylated compound. Such site(s) may be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds in mammalian cells such as CHO,

BHK,

COS). However, such sites may further be glycosylated by synthetic or semi-synthetic procedures known in the art.

Other possible modifications incude hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins- Structure andMolecule Proerties H. Freeman Co., San Francisco), pp. 79-86 (1983).

Compounds of the present invention may be changed at the DNA level, as well. The DNA sequence of any portion of the compound may be changed to codons more compatible with the chosen host cell. For Ecoli which is the preferred host cell, optimized codons are known in the art.

Codons may be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the. selected host cell. The vehicle, linker and peptide DNA sequences may be modified to include any of the foregoing sequence changes.

Methods of Making The compounds of this invention largely may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.

The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art.

-71- Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, 1i useful microbial hosts include bacteria (such as E. cli yeast (such as Saccharomvces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art.

The compounds may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Poleptides pp. 3 3 5 6 1 (Katsoyannis and Panayotis eds.); Merrifield (1963), A. Chem. Soc. 85: 2149; Davis et al.

(1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Snthess U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteis (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is-the most cost-effective method of making small peptides.

-72- Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques.

-73- Uses of the Compounds In eneral. The compounds of this invention have pharmacologic activity resulting from their ability to bind to proteins of interest as agonists, mimetics or antagonists of the native ligands of such proteins of interest. The utility of specific compounds is shown in Table 2. The activity of these compounds can be measured by assays known in the art. For the TPO-mimetic and EPO-mimetic compounds, in vivo assays are further described in the Examples section herein.

In addition to therapeutic uses, the compounds of the present invention are useful in diagnosing diseases characterized by dysfunction of their associated protein of interest. In one embodiment, a method of detecting in a biological sample a protein of interest a receptor) that is capable of being activated comprising the steps of: contacting the sample with a compound of this invention; and detecting activation of the protein of interest by the compound. The biological samples include tissue specimens, intact cells, or extracts thereof. The compounds of this invention may be used as part of a diagnostic kit to detect the presence of their associated proteins of interest in a biological sample. Such kits employ the compounds of the invention having an attached label to allow 2 0 for detection. The compounds are useful for identifying normal or abnormal proteins of interest. For the EPO-mimetic compounds, for example, presence of abnormal protein of interest in a biological sample may be indicative of such disorders as Diamond Blackfan anemia, where it is believed that the EPO receptor is dysfunctional.

Therapeutic uses of EPO-mimetic compounds. The EPO-mimetic compounds of the invention are useful for treating disorders characterized by low red blood cell levels. Included in the invention are methods of modulating the endogenous activity of an EPO receptor in a mammal, preferably methods of increasing the activity of an EPO receptor. In -74general, any condition treatable by erythropoietin, such as anemia, may also be treated by the EPO-mimetic compounds of the invention. These compounds are administered by an amount and route of delivery that is appropriate for the nature and severity of the condition being treated and may be ascertained by one skilled in the art. Preferably, administration is by injection, either subcutaneous, intramuscular, or intravenous.

Therapeutic uses of TPO-mimetic compounds. For the TPOmimetic compounds, one can utilize such standard assays as those described in W095/26746 entitled "Compositions and Methods for Stimulating Megakaryocyte Growth and Differentiation". In vivo assays also appear in the Examples hereinafter.

The conditions to be treated are generally those that involve an existing megakaryocyte/platelet deficiency or an expected megakaryocyte/platelet deficiency because of planned surgery or platelet donation). Such conditions will usually be the result of a deficiency (temporary or permanent) of active Mpl ligand in vivo. The generic term for platelet deficiency is thrombocytopenia, and hence the methods and compositions of the present invention are generally available for treating thrombocytopenia in patients in need thereof.

Thrombocytopenia (platelet deficiencies) may be present for various reasons, including chemotherapy and other therapy with a variety of drugs, radiation therapy, surgery, accidental blood loss, and other specific disease conditions. Exemplary specific disease conditions that involve thrombocytopenia and may be treated in accordance with this invention are: aplastic anemia, idiopathic thrombocytopenia, metastatic tumors which result in thrombocytopenia, systemic lupus erythematosus, splenomegaly, Fanconi's syndrome, vitamin B12 deficiency, folic acid deficiency, May-Hegglin anomaly, Wiskott-Aldrich syndrome, and paroxysmal nocturnal hemoglobinuria. Also, certain treatments for AIDS result in thrombocytopenia AZT). Certain wound healing disorders might also benefit from an increase in platelet numbers.

With regard to anticipated platelet deficiencies, due to future surgery, a compound of the present invention could be administered several days to several hours prior to the need for platelets. With regard to acute situations, accidental and massive blood loss, a compound of this invention could be administered along with blood or purified platelets.

The TPO-mimetic compounds of this invention may also be useful in stimulating certain cell types other than megakaryocytes if such cells are found to express Mpl receptor. Conditions associated with such cells that express the Mpl receptor, which are responsive to stimulation by the Mpl ligand, are also within the scope of this invention.

The TPO-mimetic compounds of this invention may be used in any situation in which production of platelets or platelet precursor cells is desired, or in which stimulation of the c-Mpl receptor is desired. Thus, for example, the compounds of this invention may be used to treat any condition in a mammal wherein there is a need of platelets, megakaryocytes, and the like. Such conditions are described in detail in the following exemplary sources: W095/26746; W095/21919; W095/18858; WO95/21920 and are incorporated herein.

The TPO-mimetic compounds of this invention may also be useful in maintaining the viability or storage life of platelets and/or megakaryocytes and related cells. Accordingly, it could be useful to include an effective amount of one or more such compounds in a composition containing such cells.

The therapeutic methods, compositions and compounds of the present invention may also be employed, alone or in combination with other cytokines, soluble Mpl receptor, hematopoietic factors, interleukins, growth factors or antibodies in the treatment of disease states -76characterized by other symptoms as well as platelet deficiencies. It is anticipated that the inventive compound will prove useful in treating some forms of thrombocytopenia in combination with general stimulators of hematopoiesis, such as IL-3 or GM-CSF. Other megakaryocytic stimulatory factors, meg-CSF, stem cell factor (SCF), leukemia inhibitory factor (LIF), oncostatin M (OSM), or other molecules with megakaryocyte stimulating activity may also be employed with Mpl ligand. Additional exemplary cytokines or hematopoietic factors for such co-administration include IL-1 alpha, IL- beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha), consensus interferon, IFN-beta, or IFN-gamma. It may further be useful to administer, either simultaneously or sequentially, an effective amount of a soluble mammalian Mpl receptor, which appears to have an effect of causing megakaryocytes to fragment into platelets once the megakaryocytes have reached mature form. Thus, administration of an inventive compound (to enhance the number of mature megakaryocytes) followed by administration of the soluble Mpl receptor (to inactivate the ligand and allow the mature megakaryocytes to produce platelets) is 2 0 expected to be a particularly effective means of stimulating platelet production. The dosage recited above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by conventional methods.

In cases where the inventive compounds are added to compositions of platelets and/or megakaryocytes and related cells, the amount to be included will generally be ascertained experimentally by techniques and assays known in the art. An exemplary range of amounts is-0.1 lg-1 mg inventive compound per 10 6 cells.

-77- Pharmaceutical Compositions hi General. The present invention also provides methods of using pharmaceutical compositions of the inventive compounds. Such pharmaceutical compositions may be for administration for injection, or for oral, pulmonary, nasal, transdermal or other forms of administration. In general, the invention encompasses pharmaceutical compositions comprising effective amounts of a compound of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content Tris-HC1, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents Tween Polysorbate 80), anti-oxidants ascorbic acd, sodium metabisulfite), preservatives Thimersol, benzyl alcohol) and bulking substances lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,

PA

18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.

Oral dosae forms. Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89 of Remint n Phariaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton

PA

18042, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, -78liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers U.S. Patent No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Chapter 10 of Marshall, Modem Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, herein incorporated by reference. In general, the formulation will include the inventive compound, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the above inventive compounds. If necessary, the compounds may be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits inhibition of proteolysis; and uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached vehicles in this invention may also be used for this purpose.

Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY,, pp 367- 83; Newmark, et al. (1982), T. Appl. Biochem. 4:185-9. Other polymers that couldbe used are poly-1,3-dioxolane and poly-l,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.

-79- For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See US Patent No. 5,792,451, "Oral drug delivery composition and methods".

The compounds of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the compound of the invention with an inert material. These, diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not liinited to starch including the commercial disintegrantased on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange

I

peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent.

Surfactants may include anionic detergents such as sodiumiauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or -81benzethonium chloride. The list of potential nonionic detergents that could-be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

Additives may also be included in the formulation to enhance uptake of the compound. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The compound of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms gums. Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid.

-82- A mix of materials might be used to provide the optimum film coating. Filtn coating may be carried out in a pan coater or in a fluidized bed or by compression coating.

Pulmonary delivery forms. Also contemplated herein is.pulmonary delivery of the present protein (or derivatives thereof). The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al.

(1990), Internatl. i. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al. (1989), T. Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146 (endothelin- Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (al-antitrypsin); Smith et al. (1989), 1. Clin. Invest. 84:1145-6 (al-proteinase); Oswein et al. (March 1990), "Aerosolization- ofProteins'-Proc-Symp-Resp -Drug-DeliveryilI Keystone, Colorado (recombinant human growth hormone); Debs et al.

(1988), 1. Immunol. 140: 3482-8 (interferon-y and tumor necrosis factor a) and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulnonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; ahd'he Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.

-83- All such devices require the use of formulations suitable for the dispensing ofthe inventive compound. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.

The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than pm (or microns), most preferably 0.5 to 5 am, for most effective delivery to the distal lung.

r Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

2 0 Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a buffer and a simple sugar for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive -84compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, 50 to 90% by weight of the formulation.

Nasal delivery forms. Nasal delivery of the inventive compound is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.

Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated.

Dosages. The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.

Specific preferred embodiments The inventors have determined preferred peptide sequences for molecules having many different kinds of activity. The inventors have further determined preferred structures of these preferred peptides combined with preferred linkers and vehicles. Preferred structures for these preferred peptides listed in Table 21 below.

Table 21-Preferred embodiments Sequence/structure SEQ Activity

ID

EGPTLRQWLAARA-(G),AEGPTLRQWLAARA 3137 TPO-mimnetic IEGPTLRQWLAARA(G)AEGPTLRQWLAARA-(GX, F' 338 TPO-mimnetic 5 -IEGPTLRQWLAARA TPO-Mimnetic 1032 IEGPTLRQWLAARA

-G

5 F'TPO-mimetic 1033 F'..(G),-GGTYSCHFGPLTWVCKPQGG-(G) 4 339 EPO-mimnetic GGTYSCHFGPLTWVCKPQGG GGTYSCHFGPLTWVCKPQGG-(G) 4 EPO-mhimetic GGTY SCHFGPLTWVCKPOGG-(G)-F' GGTYSCHFGPLTWVCKPQGG-(G) 5 EPO-mimetic 1034 F'-(G),-DFLPHYKNTSLGHRP TNF-a inhibitor 1045 DFLPYKNTLGHR-(G)-FlTNF-a inhibitor DFLPYKNTLGHR-(G)-F'1046 FEWVTPGYWQPYALPL IL-i R antagonist 1047 FEWTGYWQYALP-(G)-F'IL-i R antagonist FEWTGYWQYALP-(G)-F'1048 -VEPNCDIHVMWEWECFERL 1049

VEPNCDIH-VMWEWECFERL-(GW-

1050

F'-(G),-CTTHWGFTLC

1051

OTTHWGFTLC-(G)-F

1 1052 is an Fc domain as defined previously herein.

Working examples M MP inhibitor MMP inhibitor -86- The compounds described above may be prepared as described below. These examples comprise preferred embodiments of the invention and are illustrative rather than limiting.

Example 1 TPO-Mimetics The following example uses peptides identified by the numbers appearing in Table A hereinafter.

Preparation of peptide 19. Peptide 17b (12'mg) and MeO-PEG-SH 5000 (30 mg, 2 equiv.) were dissolved in 1 ml aqueous buffer (pH The mixture was incubated at RT for about 30 minutes and the reaction was checked by analytical HPLC, which showed a 80% completion of the reaction. The pegylated material was isolated by preparative HPLC.

Preparation of peptide 20. Peptide 18 (14 mg) and MeO-PEGmaleimide (25 mg) were dissolved in about 1.5 ml aqueous buffer (pH 8).

The mixture was incubated at RT for about 30 minutes, at which time about 70% transformation was complete as monitored with analytical HPLC by applying an aliquot of sample to the HPLC column. The pegylated material was purified by preparative HPLC.

Bioactivity assay. The TPO in vitro bioassay is a mitogenic assay utilizing an IL-3 dependent clone of murine 32D cells that have been transfected with human mpl receptor. This assay is described in greater detail in WO 95/26746. Cells are maintained in MEM medium containing Fetal Clone II and 1 ng/ml mIL-3. Prior to sample addition, cells are prepared by rinsing twice with growth medium lacking mIL-3. An extended twelve point TPO standard curve is prepared, ranging from 33 to 39 pg/ml. Four dilutions, estimated to fall within the linear portion of the standard curve, (100 to 125 pg/ml), are prepared for each sample and run in triplicate. A volume of 100 ul of each dilution of sample or standard is added to appropriate wells of a 96 well microtiter plate -87containing 10,000 cells/well. After forty-four hours at 37 OC and 10% CO, MTS (a tetrazolium compound which is bioreduced by cells to a formazan) is added to each well. Approximately six hours later, the optical density is read on a plate reader at 490 nm. A dose response curve (log TPO concentration vs. Background) is generated and linear regression analysis of points which fall in the linear portion of the standard curve is performed. Concentrations of unknown test samples are determined using the resulting linear equation and a correction for the dilution factor.

TMP tandeme s cine linkers. Our design of sequentially linked TMP repeats was based on the assumption that a dimeric form of TMP was required for its effective interaction with c-Mpl (the TPO receptor) and that depending on how they were wound up against each other in the receptor context, the two TMP molecules could be tethered together in the C- to N-terminus configuration in a way that would not perturb the global dimeric conformation. Clearly, the success of the design of tandem linked repeats depends on proper selection of the length and composition of the linker that joins the C- and N-termini of the two sequentially aligned TMP monomers. Since no structural information of the TMP bound to c-Mpl was available, a series of repeated peptides with linkers composed of 0 to 10 and 14 glycine residues (Table A) were synthesized. Glycine was chosen because of its simplicity and flexibility, based on the rationale that a flexible polyglycine peptide chain might allow for the free folding of the two tethered TMP repeats into the required conformation, while other amino acid sequences may adopt undesired secondary structures whose rigidity might disrupt the correct packing of the repeated peptide in the receptor context.

The resulting peptides are readily accessible by crinentional solid phase peptide synthesis methods (Merrifield (1963), I Amer. hem. So 2149) with either Fmoc or t-Boc chemistry. Unlike the synthesis of the -88- C-terminally linked parallel dimer which required the use of an orthogonally protected lysine residue as the initial branch point to build the two peptide chains in a pseudosymmetrical way (Cwirla et al. (1997), Science 276: 1696-9), the synthesis of these tandem repeats was a straightforward, stepwise assembly of the continuous peptide chains from the C- to N-terminus. Since dimerization of TMP had a more dramatic effect on the proliferative activity than binding affinity as shown for the Cterminal dimer (Cwirla et al. (1997)), the synthetic peptides were tested directly for biological activity in a TPO-dependent cell-proliferation assay using an IL-3 dependent clone of murine 32D cells transfected with the full-length c-Mp (Palacios et al., Cell 41:727 (1985)). As the test results showed, all the polyglycine linked tandem repeats demonstrated >1000 fold increases in potency as compared to the monomer, and were even more potent than the C-terminal dimer in this cell proliferation assay. The absolute activity of the C-terminal dimer in our assay was lower than that of the native TPO protein, which is different from the previously reported findings in which the C-terminal dimer was found to be as active as the natural ligand (Cwirla et al. (1997)). This might be due to differences in the conditions used in the two assays. Nevertheless, the difference in activity between tandem (C terminal of first monomer linked to N terminal of second monomer) and C-terminal (C terminal of first monomer linked to C terminal of second monomer; also referred to as parallel) dimers in the same assay clearly demonstrated the superiority of tandem repeat strategy over parallel peptide dimerization. It is interesting to note that a wide range of length is tolerated by the linker. The optimal linker between tandem peptides with the selected TMP monomers apparently is composed of 8 glycines.

Other tandem repeats. Subsequent to this first series of TMP tandem repeats, several other molecules were designed either with -89different linkers or containing modifications within the monomer itself.

The first of these molecules, peptide 13, has a linker composed of GPNG, a sequence known to have a high propensity to form a p-turn-type secondary structure. Although still about 100-fold more potent than the monomer, this peptide was found to be >10-fold less active than the equivalent GGGG-linked analog. Thus, introduction of a relatively rigid p-turn at the linker region seemed to have caused a slight distortion of the optimal agonist conformation in this short linker form.

The Trp9 in the TMP sequence is a highly conserved residue among the active peptides isolated from random peptide libraries. There is also a highly conserved Trp in the consensus sequences of EPO mimetic peptides and this Trp residue was found to be involved in the formation of a hydrophobic core between the two EMPs and contributed to hydrophobic interactions with the EPO receptor. Livnah et al. (1996), Science 273: 464- 71). By analogy, the Trp9 residue in TMP might-have a similar function in dimerization of the peptide ligand, and as an attempt to modulate and estimate the effects of noncovalent hydrophobic forces exerted by the two indole rings, several analogs were made resulting from mutations at the Trp. So in peptide 14, the Trp residue was replaced in each of the two TMP monomers with a Cys, and an intramolecular disulfide bond was formed between the two cysteines by oxidation which was envisioned to mimic the hydrophobic interactions between the two Trp residues in peptide dimerization. Peptide 15 is the reduced form of peptide 14. In peptide 16, the two Trp residues were replaced by Ala. As the assay data show, all three analogs were inactive. These data further demonstrated that Trp is critical for the activity of the TPO mimetic peptide, not just for dimer formation.

The next two peptides (peptide 17a, and 18) each contain in their 8amino acid linker a Lys or Cys residue. These two compounds are precursors to the two PEGylated peptides (peptide 19 and 20) in which the side chain of the Lys or Cys is modified by a PEG moiety. A PEG moiety was introduced at the middle of a relatively long linker, so that the large PEG component (5 kDa) is far enough away from the critical binding sites in the peptide molecule. PEG is a known biocompatible polymer which is increasingly used as a covalent modifier to improve the pharmacokinetic profiles of peptide- and protein-based therapeutics.

A modular, solution-based method was devised for convenient PEGylation of synthetic or recombinant peptides. The method is based on the now well established chemoselective ligation strategy which utilizes the specific reaction between a pair of mutually reactive functionalities.

So, for pegylated peptide 19, the lysine side chain was preactivated with a bromoacetyl group to give peptide 17b to accommodate reaction with a thiol-derivatized PEG. To do that, an orthogonal protecting group, Dde, was employed for the protection of the lysine e-amine. Once the whole peptide chain was assembled, the N-terminal amine was reprotected with t-Boc. Dde was then removed to allow for the bromoacetylation. This strategy gave a high quality crude peptide which was easily purified using conventional reverse phase HPLC. Ligation of the peptide with the thiolmodified PEG took place in aqueous buffer at pH 8 and the reaction completed within 30 minutes. MALDI-MS analysis of the purified, pegylated material revealed a characteristic, bell-shaped spectrum with an increment of 44 Da between the adjacent peaks. For PEG-peptide 20, a cysteine residue was placed in the linker region and its side chain thiol group would serve as an attachment site for a maleimide-containing

PEG.

Similar conditions were used for the pegylation of this peptide. As the assay data revealed, these two pegylated peptides had everr higher in vitro bioactivity as compared to their unpegylated counterparts.

-91- Peptide 21 has in its 8-amino acid linker a potential glycosylation motif,-NGS. Since our exemplary tandem repeats are made up of natural amino acids linked by peptide bonds, expression of such a molecule in an appropriate eukaryotic cell system should produce a glycopeptide with the carbohydrate moiety added on the side chain carboxyamide of Asn.

Glycosylation is a common post-translational modification process which can have many positive impacts on the biological activity of a given protein by increasing its aqueous solubility and in vivo stability. As the assay data show, incorporation of this glycosylation motif into the linker maintained high bioactivity. The synthetic precursor of the potential glycopeptide had in effect an activity comparable to that of the linked analog. Once glycosylated, this peptide is expected to have the same order of activity as the pegylated peptides, because of the similar chemophysical properties exhibited by a PEG and a carbohydrate moiety.

The last peptide is a dimer of a tandem repeat. It was prepared by oxidizing peptide 18, which formed an intermolecular disulfide bond between the two cysteine residues located at the linker. This peptide was designed to address the possibility that TMP was active as a tetramer. The assay data showed that this peptide was not more active than an average tandem repeat on an adjusted molar basis, which indirectly supports the idea that the active form of TMP is indeed a dimer, otherwise dimerization of a tandem repeat would have a further impact on the bioactivity.

In order to confirm the in vitro data in animals, one pegylated

TMP

tandem repeat (compound 20 in Table A) was delivered subcutaneously to normal mice via osmotic pumps. Time and dose-dependent increases were seen in platelet numbers for the duration of treatment. Peak platelet levels over 4-fold baseline were seen on day 8. A dose of 10-Tg/kg/day of the pegylated TMP repeat produced a similar response to rHuMGDF (non-pegylated) at 100 pg/kg/day delivered by the same route.

-92- Table A-TPO-mimetic Peptides Peptide Compound -SEQ ID -Relative No. NO: Potency a rn TMP monomer TMP C-C dimer

TMP-(G),-T

1 2 3 4 6 7 8 9 11 12 13 14

MP:

n=0 n I n=2 n =3 n=4 n n 6 n=7 n=8 n=9 n =110 n= 14

TMP-GPNG-TMP

lEGPTLRQ-GLAARA-GGGGGGGG-IEGPTLRO.LAARA (cyclic 4+4+ 4+4+ 4+4+ 44+4-.

4+4+ 4+4+ 4+4 lEG PTLRQLAARA-GGGGGGGG- IEG PTLRQLAARA (linear) 16 lEG PTLRQALAARA-GGGGGGGG-

IEGPTLRQALAARA

17la TMP-GGGKGGGG-TMP 17lb TMP.GGGK(BrAc)GGGG-TMP 18 TMP-GGGCGGGG-TMP 19 TMP-GGGK(PEG)GGGG-TMP

TMP-GGGC(PEG)GGGG-TMP

21 TMP-.GGGN*GSGG-TMP 22- TMP-GGGCGGGG-TMP

TMP-GGGCGGGG-TMP

357 358 359.

360 361 362 363- +4+4

ND

4+4+ +4+4+ 4+4+ +4.4+ 363 -93- Discussion. It is well accepted that MGDF acts in a way similar to hGH, one molecule of the protein ligand binds two molecules of the receptor for its activation. Wells et al.(1996), Ann. Rev. Biochem. 65: 609- 34. Now, this interaction is mimicked by the action of a much smaller peptide, TMP. However, the present studies suggest that this mimicry requires the concerted action of two TMP molecules, as covalent dimerization of TMP in either a C-C parallel or C-N sequential fashion increased the in vitro biological potency of the original monomer by a factor of greater than 10'. The relatively low biopotency of the monomer is probably due to inefficient formation of the noncovalent dimer. A preformed covalent repeat has the ability to eliminate the entropy barrier for the formation of a noncovalent dimer which is exclusively driven by weak, noncovalent interactions between two molecules of the small, 14residue peptide.

It is intriguing that this tandem repeat approach had a similar effect on enhancing bioactivity as the reported C-C dimerization is intriguing.

These two strategies brought about two very different molecular configurations. The C-C dimer is a quasi-symmetrical molecule, while the tandem repeats have no such symmetry in their linear structures. Despite this difference in their primary structures, these two types of molecules appeared able to fold effectively into a similar biologically active conformation and cause the dimerization and activation of c-Mpl. These experimental observations provide a number of insights into how the two TMP molecules may interact with one another in binding to c-Mpl. First, the two C-termini of the two bound TMP molecules must be in relatively close proximity with each other, as suggested by data on the C-terminal dimer. Second, the respective N- and C-termini of the two TMP molecules in the receptor complex must also be very closely aligned with each other, such that they can be directly tethered together with a single peptide bond -94to realize the near maximum activity-enhancing effect brought about by the tandem repeat strategy. Insertion of one or more (up to 14) glycine residues at the junction did not increase (or decrease) significantly the activity any further. This may be due to the fact that a flexible polyglycine peptide chain is able to loop out easily from the junction without causing any significant changes in the overall conformation. This flexibility seems to provide the freedom of orientation for the TMP peptide chains to fold into the required conformation in interacting with the receptor and validate it as a site of modification. Indirect evidence supporting this came from the study on peptide 13, in which a much more rigid b-turnforming sequence as the linker apparently forced a deviation of the backbone alignment around the linker which might have resulted in a slight distortion of the optimal conformation, thus resulting in a moderate decrease in activity as compared with the analogous compound with a 4-Gly linker. Third, Trp9 in TMP plays a similar role as Trpl3 in EMP, which is involved not only in peptide:peptide interaction for the formation of dimers but also is important for contributing hydrophobic forces in peptide:receptor interaction. Results obtained with the W to C mutant analog, peptide 14, suggest that a covalent disulfide linkage is not sufficient to approximate the hydrophobic interactions provided by the Trp pair and that, being a short linkage, it might bring the two TMP monomers too close, therefore perturbing the overall conformation of the optimal dimeric structure.

An analysis of the possible secondary structure of the TMP peptide can provide further understanding on the interaction between TMP and c- Mpl. This can be facilitated by making reference to the reported structure of the EPO mimetic peptide. Livnah et al. (1996), Science 273:464-75 The receptor-bound EMP has a b-hairpin structure with a b-turn formed by the highly consensus Gly-Pro-Leu-Thr at the center of its sequence. Instead of GPLT, TMP has a highly selected GPTL sequence which is likely to form a similar.turn.- However, this turn-like motif is located near the N-terminal part in TMP. Secondary structure prediction using Chau-Fasman method suggests that the C-terminal half of the peptide has a tendency to adopt a helical conformation. Together with the highly conserved Trp at position 9, this C-terminal helix may contribute to the stabilization of the dimeric structure. It is interesting to note that most of our tandem repeats are more potent than the C-terminal parallel dimer. Tandem repeats seem to give the molecule a better fit conformation than does the C-C parallel dimerization. The seemingly asymmetric feature of a tandem repeat might have brought it closer to the natural ligand which, as an asymmetric molecule, uses two different sites to bind two identical receptor molecules.

Introduction of a PEG moiety was envisaged to enhance the in vivo activity of the modified peptide by providing it a protection against proteolytic degradation and by slowing down its clearance through renal filtration. It was unexpected that pegylation could further increase the in vitro bioactivity of a tandem repeated TMP peptide in the cell-based proliferation assay.

Example 2 Fc-TMP fusions TMPs (and EMPs as described in Example 3) were expressed in either monomeric or dimeric form as either N-terminal or C-terminal fusions to the Fc region of human IgG1. In all cases, the expression construct utilized the luxPR promoter promoter in the plasmid expression vector pAMG21.

Fc-TMP. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of the TPO-mimetic peptide Was constructed using standard PCR technology. Templates for PCR reactions were the pFc-A3 vector and a synthetic TMP gene. The synthetic gene was -96constructed from the 3 overlapping oligonucleotides (SEQ ID NOS: 364, 365, and 366; respectively) shown below: 1842-97 AAA AAA GGA TCC TCG AGA TTA AGC ACG AGC AGC CAG CCA CTG ACG CAG AGT CGG ACC 1842-98 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCG ACT CTG CGT 1842-99 CAG TGG CTG GCT GCT CGT GCT TAA TCT CGA GGA TCC TTT

TTT

These oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 367 and 368, respectively) shown below:

AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT

1

CCAGGCTGAGACGCAGTCACCGACCGACGAGCACGA

a K G G G G I E G P TL R Q W L A A R A

TAATCTCGAGGATCCTTTTTT

61 81

ATTAGAGCTCCTAGGAAAAAA

a This duplex was amplified in a PCR reaction using 1842-98 and 1842-97 as the sense and antisense primers.

The Fc portion of the molecule was generated in a PCR reaction with pFc-A3 using the primers shown below (SEQ ID NOS: 369 and 370): 1216-52 AAC ATA AGT ACC TGT AGG ATC G 1830-51 TTCGATACCA CCACCTCCAC CTTTACCCGG AGACAGGGAG AGGCTCTTCTGC The oligonucleotides 1830-51 and 1842-98 contain an overlap of 24 nucleotides, allowing the two genes to be fused together in the correct reading frame by combining the above PCR products in a third reaction using the outside primers, 1216-52 and 1842-97.

The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases Xbal and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the -97gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3728.

The nucleotide and amino acid sequences (SEQ ID NOS: 5 and 6) of the fusion protein are shown in Figure 7.

Fc-TMP-TMP. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a dimer of the TPO-mimetic peptide was constructed using standard PCR technology. Templates for PCR reactions were the pFc-A3 vector and a synthetic TMP-TMP gene. The synthetic gene was constructed from the 4 overlapping oligonucleotides (SEQ ID NOS: 371 to 374, respectively) shown below: 1830-52 AAA GGT GGA GGT GGT GGT ATC GAA GGT CCG ACT CTG CGT CAG TGG CTG GCT GCT CGT GCT 1830-53 ACC TCC ACC ACC AGC ACG AGC AGC CAG CCA CTG ACG CAG AGT CGG ACC 1830-54 GGT T GT GGA GGT GGC GGC GGA GGT ATT GAG GGC CCA ACC CTT CGC CAA TGG CTT GCA GCA CGC GCA 1830-55 AAA AAA AGG ATC CTC GAG ATT ATG CGC GTG CTG CAA GCC ATT GGC GAA GGG TTG GGC CCT CAA TAC CTC CGC CGC C The 4 oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 375 and 376, respectively) shown below:

AAAGGTGGAGGTGGTGGTATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGCTCGTGCT

1 CCAGGCTGAGACGCA GTCATCCGACCGACGAGCACGA a KG G G G G IE G P T L R Q W LAARA

GGTGGTGGAGGTGGCGGCGGAGGTATTGAGGCCCAACCCTTCCCAATGGCTTGCAGCA

+120

CCACCACCTCCACCGCCCTCCATAACTCCCGGGTTGGGAAGCGGTTACCGAACGTCGT

a G G G G G G G G I EG P TL R Q W LA A

CGCGCA

GCGCGTATTAGAGCTCCTAGGAAAAAAA

a R A This duplex was amplified in a PCR reaction using 1830-52 and 1830-55 as the sense and antisense primers.

The Fe portion of the molecule was generated in a PCR reaction with pFc-A3 using the primers 1216-52 and 1830-51 as described above for -98- Fc-TMP. The full length fusion gene was obtained from a third PCR reaction using the outside primers 1216-52 and 1830-55.

The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described in example 1. Clones Were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3727.

The nucleotide and amino acid sequences (SEQ ID NOS: 7 and 8) of the fusion protein are shown in Figure 8.

TMP-TMP-Fc. A DNA sequence coding for a tandem repeat of the TPO-mimetic peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. Templates for PCR reactions were the EMP-Fc plasmid from strain #3688 (see Example 3) and a synthetic gene encoding the TMP dimer. The synthetic gene for the tandem repeat was constructed from the 7 overlapping oligonucleotides shown below (SEQ ID NOS: 377 to 383, respectively): 1885-52 TTT TTT CAT ATG ATC GAA GGT CCG ACT CTG CGT CAG TGG 1885-53 AGC ACG AGC AGC CAG CCA CTG ACG CAG AGT CGG ACC TTC GAT CAT ATG 1885-54 CTG GCT GCT CGT GCT T G GGA GGC GGT GGG GAC AAA ACT CAC ACA 1885-55 CTG GCT GCT CGT GCT GGC GGT GGT GGC GGA GGG GGT GGC ATT GAG GGC CCA 1885-56 AAG CCA TTG GCG AAG GGT TGG GCC CTC AAT GCC ACC CCC TCC GCC ACC ACC GCC 1885-57 ACC CTT CGC CAA TGG CTT GCA GCA CGC GCA GGG GGA GGC GGT GGG GAC AAA ACT 1885-58 CCC ACC GCC TCC CCC TGC GCG TGC TGC These oligonucleotides were annealed to form the duplex shown encoding an amino acid sequence shown below (SEQ ID NOS 384 and 385): -99- TTTTTTCATATGATCGAAGGTCCGACTCTGCGTCAGTGGCTGGCTGC

TCGTGCTGGCGGT

GTATACTAGCCCAGGCTGAGACGCAGTCACCGACCGACGAGCACGACCGCCA

M I E G P T L R Q W L A A R A G G

GGTGGCGGAGGGGGTGGCATTGAGGGCCCAACCCTTCGCCTGGCGCTGTCGTGCTGTTT

61 120

CCACCGCCTCCCCCACCGTAACTCCCGGGTTGGAAGCGGTTACCGAACGTCGTGCGCGT

a G G G G G G I E G P T L R Q W L A A R A 121 C.CCTC 180 CCCCCTCCGCCACCC A G G G G G D K

ACTCACACA

181 189 a T HT 2 This duplex was amplified in a PCR reaction using 1885-52 and 1885-58 as the sense and antisense primers.

The Fc portion of the molecule was generated in a PCR reaction with DNA from the EMP-Fc fusion strain #3688 (see Example 3) using the primers 1885-54 and 1200-54. The full length fusion gene was obtained from a third PCR reaction using the outside primers 1885-52 and 1200-54.

The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. co strain 3 0 2596 cells as described for Fc-EMP herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3798.

The nucelotide and amino acid sequences (SEQ ID NOS: 9 and of the fusion protein are shown in Figure 9.

TMP-Fc. A DNA sequence coding for a monomer of the TPOmimetic peptide fused in-frame to the Fc region of human IgGI was obtained fortuitously in the ligation in TMP-TMP-Fc, presumably due to the ability of primer 1885-54 to anneal to 1885-53 as well as to 1885-58.

A

4 0 single clone having the correct nucleotide sequence for the TMP-Fc construct was selected and designated Amgen strain #3788.

-100- The nucleotide and amino acid sequences (SEQ ID NOS: 11 and 12) of the fusion protein are shown in Figure Expression in E. coli. Cultures of each of the pAMG21-Fc-fusion constructs in E. coli GM221 were grown at 37 °C in Luria Broth medium containing 50 mg/ml kanamycin. Induction of gene product expression from the luxPR promoter was achieved following the addition of the synthetic autoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a final concentration of 20 ng/ml. Cultures were incubated at 37 °C for a further 3 hours. After 3 hours, the bacterial cultures were examined by microscopy for the presence of inclusion bodies and were then collected by centrifugation. Refractile inclusion bodies were observed in induced cultures indicating that the Fc-fusions were most likely produced in the insoluble fraction in E. coli. Cell pellets were lysed directly by resuspension in Laemmli sample buffer containing 10% b-mercaptoethanol and were analyzed by SDS-PAGE. In each case, an intense coomassie-stained band of the appropriate molecular weight was observed on an SDS-PAGE gel.

pAMG21. The expression plasmid pAMG21 can be derived from the Amgen expression vector pCFM1656 (ATCC #69576) which in turn be derived from the Amgen expression vector system described in US Patent No. 4,710,473. The pCFM1656 plasmid can be derived from the described pCFM836 plasmid (Patent No. 4,710,473) by: destroying the two endogenous NdeI restriction sites by end filling with T4 polymerase enzyme followed by blunt end ligation; replacing the DNA sequence between the unique AatII and Clal restriction sites containing the synthetic PL prom-ter with a similar fragment obtained from pCFM636 (patent No. 4,710,473) containing the PL promoter (see SEQ ID NO: 386 below); and -101substituting the small DNA sequence between the unique CIaI and I 2 pLrestrition sites with the oligonucleotide having the sequence of SEQ ID NO: 388.

SEQ ID NO: 386: aaII

TAAATTCATAT

5'CTAATTCCGCTCTCACC'ir~;cCAfACAATGCCCCCCTGCA A A AAATATTATTCAGTATA 3' TGCAGATTAAGGCGAGTGGATGGTTTGTTACGGGGGACG

*AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA-

-TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGT -TACCACTGGCGGTGATACTGAGCACAT 3' -ATGGTGACCGCCACTATGACTCGTGTAGC Cla' SEQ ID NO: 387: CGATTTGATTCTAGAAGGAGGAATAACATATGGTTCG AACG 3' 3' TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC Clal The expression plasmid pAMG2I can then be derived from pCFM1656 by making a series of site-directed base changes by PCR overlapping oligo mutagenesis and DNA sequence substitutions. Starting with the BglII site (plasmid bp 180) immediately 5' to the plasmid replication promoter PCopB and proceeding toward the plasmid replication genes, the base pair changes are as shown in Table B below.

-102- Table B-Base pair changes resulting in pAMG21 pAM2 b bp in pCM 1656i b12 changed to in 12AMG21 204 428 509 617 679 980 994 1004 1007 1028 1047 1178 1466 2028 2187 2480 2499-2502

TIA

AIT

GIG

GIG

TIA

GIG

AIr

CG

AIT

CIG

GIG

GIG

GIG

G

CG

G/C

A/T

insert two GIG. bp

T/A

CG

AIT,

CG

T/A

T/A

T/A

T/A

T/A

bp deletion

T/A

T/A

TCAC

MIA

CAGT

2642 3435.

3446 3643

ICCAGC

AGGOTOG

7 bp deletion

GIG

GIC

AfT -103- The DNA sequence between the unique AatlI (position #4364 in pCFM1656) and SacI (position #4585 in pCFM1656) restriction sites is substituted with the DNA sequence (SEQ ID NO: 23) shown in Figures 17A and 17B. During the ligation of the sticky ends of this substitution DNA sequence, the outside Aatll and SacDI sites are destroyed. There are unique AatI and SaII sites in the substituted DNA.

GM221 (Amgen #2596). The Amgen host strain #2596 is an E.coli K- 12 strain derived from Amgen strain #393. It has been modified to contain both the temperature sensitive lambda repressor cl857s7 in the early ebg region and the lacl repressor in the late ebg region (68 minutes). The presence of these two repressor genes allows the use of this host with a variety of expression systems, however both of these repressors are irrelevant to the expression from luxP,. The untransformed host has no antibiotic resistances.

The ribosome binding site of the c1857s7 gene has been modified to include an enhanced RBS. It has been inserted into the eb operon between nucleotide position 1170 and 1411 as numbered in Genbank accession number M64441GbBa with deletion of the intervening ebg sequence. The sequence of the insert is shown below with lower case letters representing the ebg sequences flanking the insert shown below (SEQ ID NO: 388): ttattttcgtGCGGCCCACATTATCACCGCCAGAGGTACTAGTCAACACGCACGGTGTTAGAATTTAT

CCCTTGCGGTGATAGATTGAGCACATCGATTTGATTCTAGGGAGGGATAATATATGAGCACAAAAAAGAAA

CCATTAACACAAGAGCAGCTTGAGGACGCACGTCGCCTTAAAGCAATTTATGAAAAAAAGAAAAATGAACTTG

GCTTATCCCAGGAATCTGTCGCAGACAAGATGGGGATGGGG

CAGGCGTTGGTGCTTTATTTAATGGCAT

CAATGCATTAAATGCTTATAACGCCGCATTGCTTACAAAAATTCTCAAAGTTAGCGTTGAAGAATTTAGCCT

TCAATCGCCAGAGAATCTACGAGATGTATGAAGCGGTTAGTATGCAGCCGTCACTTAGAAGTGAGTATGAGTA

CCCTGTTTTTTCTCATGTTCAGGCAGGGATGTTCTCACCTAAGCTTAGAACCTTTACCAAAGGTGATGCGGAG

AGATGGGTAAGCACAACCAAAAAAGCCAGTGATTCTGCATTCTGGCTTGAGGTTGAAGGTAATTCCATGACCG

CACCAACAGGCTCCAAGCCAAGCTTTCCTGACGGAATGTTAATTCTCGTTGACCCTGAGCAGGCTGTTGAGCC

AGGTGATTTCTGCATAGCCAGACTTGGGGGTGATGAGTTTACCTTCAAGAAACTGATCAGGGATAGCGGTCAG

GTGTTTTTACAACCACTAACCCACAGTACCCAATGATCCCATGCAATGAGAGTTGTTCCGTTGGGGGAAAG

TTATCGCTAGTCAGTGGCCTGAAGAGACGTTTGGCTGATAGACTAGTGGA CCACTAGTttcttqcCC The construct was delivered to the chromosome using a recombinant phage called MMebg-cl857s7enhanced RBS #4 into F'tet/393.

After recombination and resolution only the chromosomal insert described -104above remains in the cell. It was renamed Ftet/GM1Q1. Ftet/GM1OI was then modified by the delivery of a 1acIQ construct into the bg operon between nucleotide position 2493 and 2937 as numbered in the Genbank accession number M64441Gb_Ba with the deletion of the intervening b sequence. The sequence of the insert is shown below with the lower case letters representing the egsequences flanking the insert (SE-Q ID NO: 389) shown below: ggcggaaaccGACGTCCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCA

ATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACC

GTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTC

IGAAGCGGCGATGGCGG

AGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGCTCCTGATTGGCGTTGCCAC

CTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCC

AGGGTGGCAGTGA AGGCTGAGCGAACGGTCCACTTGG

AACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGMAGCTGCCTGCAC

TAATGTTCCGGCGTTATTTCTTGA.TGTCTCTGACCAGACACCCATCAACAGTATTATTTCTCCCATGAAGAC

dGTACGCGACTGGGCG TGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAA GTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATCTCACTCGCAATCAAAT

TCAGCCGATAGC

GGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTT

CCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGC

GCGTTGGTGC GGA TATCTCGGT A GTGGATACGAC GATACCGAAGACGCTCATGT TATATC CCGCCGTAC

CACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAG

GCGGTGAAGGGCAATCAGCTGTTGCCCGTCTC-ACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAA

CCGCCTCTCCCCGCGCGTTGG.CCGATCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAGGGACA

GTAAGGTACCATAGGATCCaggcacagjga The construct was delivered. to the chromosom usn L recombinant phage called AGebg-LacIQ#5 into F'tet/GMIO1. After recombination and resolution only the chromosomal insert described above remains in the cell. It was renamed F'tet/GM221. The Ftet episome was cured from the strain using acridine orange at a concentration of j ig/ml in LB. The cured strain was identified as tetracyline sensitive and was stored as GM221.

ExpRression. Cultures of pAMG2.1-Fc-TMP-TMP in E. coli GM221 in Luria Broth medium containing 50 JLg/ml kanamycin were incubated at 37 0 C prior to in duc tion. Induction of Fc-TMP-TM4P gene product expression from the luxPR promoter was achieved following- the addition of the synthetic autoinducer N-(3-oxohexanoyl)-DL-homfoserine lactone to the culture media to a final concentration of 20 ng/ml and cultures were incubated at 37 0 C for a further 3 hours. After 3 hours, the bacterial -105cultures were examined by microscopy for the presence of inclusion bodies and were then collected by centrifugation. Refractile inclusion bodies were observed in induced cultures indicating that the Fc-TMP-TMP was most likely produced in the insoluble fraction in E. coli. Cell pellets were lysed directly by resuspension in Laemmli sample buffer containing *-mercaptoethanol and were analyzed by SDS-PAGE. An intense Coomassie stained band of approximately 30kDa was observed on an SDS-PAGE gel. The expected gene product would be 269 amino acids in length and have an expected molecular weight of about 29.5 kDa.

Fermentation was also carried out under standard batch conditions at the L scale, resulting in similar expression levels of the Fc-TMP-TMP to those obtained at bench scale.

Purification of Fc-TMP-TMP. Cells are broken in water (1/10) by high pressure homogenization (2 passes at 14,000 PSI) and inclusion bodies are harvested by centrifugation (4200 RPM in J-6B for 1 hour).

Inclusion bodies are solubilized in 6M guanidine, 50mM Tris, 8mM DTT, pH 8.7 for 1 hour at a 1/10 ratio. The solubilized mixture is diluted times into 2M urea, 50 mM tris, 160mM arginine, 3mM cysteine, pH The mixture is stirred overnight in the cold and then concentrated about 10 fold by ultafiltration. It is then diluted 3 fold with 10mM Tris, urea, pH 9. The pH of this mixture is then adjusted to pH 5 with acetic acid. The precipitate is'removed by centrifugation and the supernatant is loaded onto a SP-Sepharose Fast Flow column equilibrated in NaAc, 100 mM NaC1, pH 5(10mg/ml protein load, room temperature).

The protein is eluted off using a 20 column volume gradient in the same buffer ranging from 100mM NaCl to 500mM NaC1. The pool from the column is diluted 3 fold and loaded onto a SP-Sepharose HP column in mM NaAc, 150 mM NaC1, pH 5(10 mg/ml protein load, room temperature). The protein is eluted off using a 20 column volume gradient -106in the same buffer ranging from 150 mM NaCI to 400 mM NaCl. The peak is pooled and filtered.

Characterization of Fc-TMP activity. The following is a summary of in vivo data in mice with various compounds of this invention..

Mice: Normal female BDF1 approximately 10-12 weeks of age.

Bleed schedule: Ten mice per group treated on day 0, two groups started 4 days apart for a total of 20 mice per group. Five mice bled at each time point, mice were bled a minimum of three times a week. Mice were anesthetized with isoflurane and a total volume of 140-160 pl of blood was obtained by puncture of the orbital sinus. Blood was counted on a Technicon HIE blood analyzer running software for murine blood.

Parameters measured were white blood cells, red blood cells, hematocrit, hemoglobin, platelets, neutrophils.

Treatments: Mice were either injected subcutaneously for a bolus treatment or implanted with 7-day micro-osmotic pumps for continuous delivery. Subcutaneous injections were delivered in a volume of 0.2 ml.

Osmotic pumps were inserted into a subcutaneous incision made in the skin between the scapulae of anesthetized mice. Compounds were diluted in PBS with 0.1% BSA. All experiments included one control group, labeled "carrier" that were treated with this diluent only. The concentration of the test articles in the pumps was adjusted so that the calibrated flow rate from the pumps gave the treatment levels indicated in the graphs.

Compounds: A dose titration of the compound was delivered to mice in 7 day micro-osmotic pumps. Mice were treated with various compounds at a single dose of 100 pg/kg in 7 day osmotic pumps. Some of the same compounds were-then given to mice as a single-bolus injection.

Activity test results: The results of the activity experiments are shown in Figures 11 and 12. In dose response assays using 7-day micro- -107osmotic pumps, the maximum effect was seen with the compound of SEQ ID NO: 18 was at 100 g/kg/day; the 10 Ig/kg/day dose was about maximally active and 1 ig/kg/day was the lowest dose at which activity could be seen in this assay system. The compound at 10 lig/kg/day dose was about equally active as 100 lg/kg/day unpegylated rHu-MGDF in the same experiment.

-108- Example 3 Fc-EMP fusions Fc-EMP. A DNA sequence coding for the Fe region of human IgG1 fused in-fiame to a monomer of the EPO-mimetic peptide was constructed using standard PCR technology. Templates for PCR reactions were a vector containing the Fe sequence (pFc-A3, described in International application WO 97/23614, published July 3, 1997) and a synthetic gene encoding EPO monomer. The synthetic gene for the monomer was constructed from the 4 overlapping oligonucleotides (SEQ ID NOS: 390 to 393, respectively) shown below: 1798-2 TAT GAA AGG TGG AGG TGG TGG TGG AGG TAC TTA CTC TTG CCA CTT CGG CCC GCT GAC TTG G 1798-3 CGG TTT GCA AAC CCA AGT CAG CGG GCC GAA GTG GCA AGA GTA AGT ACC TCC ACC ACC ACC TCC ACC TTT CAT 1798-4 GTT TGC AAA CCG CAG GGT GGC GGC GGC GGC GGC GGT GGT ACC TAT TCC TGT CAT TTT 1798-5 CCA GGT CAG CGG GCC AAA ATG ACA GGA ATA GGT ACC ACC GCC GCC GCC GCC GCC ACC CTG The 4 oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 394 and 395, respectively) shown below:

TATGAAAGGTGGAGGTGGTGTGGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTG

TACTTTCCACCTCCACCACCACCTCCATGAA

TGAGTGAGCCGGGCGACTGAAC

b M K G G G G G G G TY S C H F G P L T W

GGTTTGCAAACCGCAGGGTGCGGCGGCGGCCGGTGGTACCTATTCCTGTCATTTT

133

CCAAACGTTGGCGTCCCACCGCCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTACC

b V C K P Q GG G G G G G T Y SC H F This duplex was amplified in a PCR reaction using 1798-18 GCA GAA GAG CCT CTC CCT GTC TCC GGG TAA AGG TGG AGG TGG TGG TGG AGG TAC TTA CTC T and 1798-19 CTA ATT GGA TCC ACG AGA TTA ACC ACC CTG CGG TTT GCA A 109 as the sense and axitisense primers (SEQ ID NOS: 39,6 and 397, respectively.

The Fc portion of the molecule was generated in a PCR reaction with pFc-A3 using the primers 1.2iG-52 AAC ATA AGT ACC, TGT AMO ATC G 1.79a-17 AGA G'TA AGT ACC TCC ACC A=C ACC TCC ACC TTT ACC CGG *AGA CAG GGA GAG GCT C-T2 CTG C w hich are SEQ ID NOS. 3 69 and 399, respectively. The oligonucleotides 1798-17 and 1798--18 contaih an overlap of 61 nticlevtides, allowing the two genes to be fused togethef in the correct reading frame by combiniing the above PCR products in a tird reaction using the outside primers,. 1-216-52 i-and 1798-19.

The final PCR gene product (the full length fusion gene) was *digested with restriction endonucleases XbaI and BanTHIL and then ligated into the vector pAMCG21 (described below), also digested with XbaI and BamfHI Ligated DNA was tansformed into competent host cells of IE. col strain 2596 (GNM2, described herein). Clones were screened for the abilit y to Produce the recombinant protein product and to possess the gene.

fusion having the correct nudcltide sequence.'A single such done was selected and designated Amgen strain #3718.

The nucleotide and amino acid sequence of the resulting fusion protein (SEQ ID NOS: 15 and 16) are shown. in Figure 13.

EMP-Fc.,A DNA sequence coding for a-monomer of the EPO0mimetic peptide fused inx-fr-ame to the Pc r-ego of human IgG1 was constructed using standard FCR technology. Templates for PCR reactions were the pFC-A3a vector and a synthetic gene encoding E1'O monomer.

The synthetic gene for the monomer was construczted from the 4 overlapping oligonix.ceCEtides 1798-4 and 1798-5 (above) and 1798- and 1798-7 (SEQ IIDNOS: 400 and 401, respectively) shown below: -110- 1798-6 GGC CCG CTG ACC TGG GTA TGT AAG CCA CAA GGG GGT GGG GGA GGC GGG GGG TAA TCT CGA G 1798-7 GAT CCT CGA GAT TAC CCC CCG CCT CCC CCA CCC CCT TGT GGC TTA CAT AC The 4 oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 402 and 403, respectively) shown below:

GTTTGCAAACCGCAGGGTGCGGCCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGC

1

GTCCCACCGCCCCGCCGCCGCCCCACCATGGATAAGGACAGTAAAACCG

A V CIK P Q G G G G GG G G T Y S C H F G

CCGCTGACCTGGGTATGTAAGCCACAAGGGGGTGGGGGAGGCGGGGGGTAATCTCGAG

122

GGCGACTGGACCCATACATTCGGTGTTCCCCCACCCCCTCCGCCCCCCATTAGAGCTCCTAG

A PL T W V C: K P Q G G G G G G G This duplex was amplified in a PCR reaction 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 GCC TCC CCC ACC CCC T as the sense and antisense primers (SEQ ID NOS: 404 and 405, respectively).

The Fc portion of the molecule was generated in a PCR reaction with pFc-A3 using the primers 1798-23 AGO GGG TGG GGG AGG CGG GGG GGA CAA AAC TCA CAC ATG TCC A and 1200-54 GTT ATT GCT CAG CGG TGG CA which are SEQ ID NOS: 406 and 407, respectively. The oligonucleotides 1798-22 and 1798-23 contain an overlap of 43 nucleotides, allowing the two genes to be fused together in the correct reading frame by combining the above PCR products in a third reaction using the outside primers, 1787-21 and 1200-54.

The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases Xbal and BamHI, and then ligated -111into the vector pAMG21 and transformed into competent E. coli strain 2596 cells.as described above. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3688.

The nucleotide and amino acid sequences (SEQ ID NOS: 17 and 18) of the resulting fusion protein are shown in Figure 14.

EMP-EMP-Fc. A DNA sequence coding for a dimer of the EPOmimetic peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. Templates for PCR reactions were the EMP-Fc plasmid from strain #3688 above and a synthetic gene encoding the EPO dimer. The synthetic gene for the dimer was constructed from the 8 overlapping oligonudeotides (SEQ ID NOS:408 to 415, respectively) shown below: 1869-23 TTT TTT ATC GAT TTG ATT CTA GAT TTG AGT TTT AAC TTT TAG AAG GAG GAA TAA AAT ATG 1869-48 TAA AAG TTA AAA CTC AAA TCT AGA ATC AAA TCG ATA AAA

AA

1871-72 GGA GGT ACT TAC TCT TGC CAC TTC GGC CCG CTG ACT TGG GTT TGC AAA CCG 1871-73 AGT CAG CGG GCC GAA GTG GCA AGA GTA AGT ACC TCC CAT ATT TTA TTC CTC CTT C 1871-74 CAG GGT G GGC GGC GGC GGC GGC GGT GGT ACC TAT TCC TGT CAT TTT GGC CCG CTG ACC TGG 1871-75 AAA ATG ACA GGA ATA GGT ACC ACC GCC CC C GCC GCC ACC CTG CGG TTT GCA AAC CCA 1871-78 GTA TGT AAG CCA CAA GGG GGT GGG GGA GGC GGG GGG GAC AAA ACT CAC ACA TGT CCA 1871-79 AGT TTT GTC CCC CCC GCC TCC CCC ACC CCC TTG TGG CTT ACA TAC CCA GGT CAG CGG GCC The 8 oligonucleotides were annealed to form the duplex encoding an amino acid sequence (SEQ ID NOS: 416 and 417, respectively) shown below:

TTTTTTATCGATTTGATTCTAGATTTAGTTTTAACTTTTAGAAGGAGGAATAAAATG

45 A -AAAATAGCTAAACTAAGATCTAAATGAAAATCTTCCTCCTTATTTTATAC -112-

GGAGGTACTTACTCTTGCCACTTCGGCCCGCTGACTTGGGTTTGCAAACCGCAGGGTGGC

61 120

CCTCCATGAATGAGAACGGTGAAGCCGGGCGACTGAACCCAAACGTTTGGCGTCCCACCG

a G G T Y S C H F G P L T W V C K P Q G G

GGCGGCGGCGGCGGTGGTACCTATTCCTGTCATTTTGGCCCGCTGACCTGGGTATGTAAG

121 180

CCGCCGCCGCCGCCACCATGGATAAGGACAGTAAAACCGGGCGACTGGACCCATACATTC

aG G G G G G T Y SC HF G P L T W V C K

CCACAAGGGGGTGGGGGAGGCGGGGGGGACAAAACTCACACATGTCCA

181 228

GGTGTTCCCCCACCCCCTCCGCCCCCCCTGTTTTGA

a P Q G G G G G G G D K TH TC P This duplex was amplified in a PCR reaction using 1869-23 and 1871-79 (shown above) as the sense and antisense primers.

The Fc portion of the molecule was generated in a PCR reaction with strain 3688 DNA using the primers 1798-23 and 1200-54 (shown above).

The oligonudeotides 1871-79 and 1798-23contain an overlap of 31 nucleotides, allowing the two genes to be fused together in the correct reading frame by combining the above PCR products in a third reaction using the outside primers, 1869-23 and 1200-54.

The final PCR gene product (the full length fusion gene) was digested with restriction endonucleases XbaI and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described for Fc-EMP. Clones were screened for ability to produce the recombinant protein product and possession of the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3813.

The nucleotide and amino acid sequences (SEQ ID NOS: 19 and respectively) of the resulting fusion protein are shown in Figure 15. There is a silent mutation at position 145 (A to G, shown in boldface) such that the final construct has a different nucleotide sequence than the oligonucleotide 1871-72 from which it was derived.

Fc-EMP-EMP. A DNA sequence coding for the Fc region of human IgGI fused in-frame to a dimer of the EPO-mimetic peptide was 113 constructed using standard PCR technology. Templates for PCR reactions were the plasmids from strains 3688 and 3813 above.

The Fc portion of the molecule was generated in a PCR reaction with strain 3688 DNA using the primers 1216-52 and 1798-17 (shown above). The ENMP dimer portion of the molecule was the product of a second PCR reaction with strain 3813 DNA using the primers 1798-18 (also shown above) and SEQ ID NO: 418, shown below: 1798-20 CTA AT GGA TC TCG AGA TTA ACC CCC TTG, TGG CTT ACAT The oligonucleotides 1798-17 and 1798-18 contain an overlap of 61 nudcleotides, allowing the two genes to be fused together in the correct reading frame by combining the above FCR products in a third reaction using the outside primers, 1216-52 and 1798-20.

The final PCR gene product (the full length fusion gene) was digested with restriction endonudcleases Xbal and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coi strain 2596 cells as described for Fc-EMP. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #3822.

The nudeotide and amino acid sequences (SEQ ID NOS:21 and 22 respectively) of the fusion protein are shown in Figure 16.

Characterization of Fc-EMP activity. Characterization was carried out in vivo as follows.

Mice: Normal female BDF1 approximately 10-12 weeks of age.

Bleed schedule: Ten mice per group treated on day 0, two groups started 4 days apart for a total of 20 mice per group. Five mice bled at each time point, mice were bled a maximum of three times a week. Mice were anesthetized with isoflurane and a total volume of 140-160 ml of blood was obtained by puncture of the orbital sinus. Blood was counted 114 on a Technicon HIE blood analyzer running software for murine blood.

Parameters -measured were WBC, RBC, HCT, HGB, PLT, NEUT, LYMPH.

Treatments: Mice were either injected subcutaneously for a bolus treatment or implanted with 7 day micro-osmotic pumps for continuous delivery. Subcutaneous injections were delivered in a volume of 0.2 ml.

Osmotic pumps were inserted into a subcutaneous incision made in the skin between the scapulae of anesthetized mice. Compounds were diluted in PBS with 0.1% BSA. All experiments included one control group, labeled "carrier" that were treated with this diluent only. The concentration of the test articles in the pumps was adjusted so that the calibrated flow rate from the pumps gave the treatment levels indicated in the graphs.

Experiments:.Various Fc-conjugated EPO mimetic peptides (EMPs) were delivered to mice as a single bolus injection at a dose of 100 Lg/kg..

Fc-EMPs were delivered to mice in 7-day micro-osmotic pumps'. The pumps were not replaced at the end of 7 days. Mice were bled until day 51 when HGB and HCT returned to baseline levels.

Example 4 TNF-a inhibitors Fc-TNF-a inhibitors. A DNA sequence coding for the Fc region of human IgGI fused in-frame to a monomer of the TNF-a inhibitory peptide was constructed using standard PCR technology. The Fc and 5 glycine linker portion of the molecule was generated in a PCR reaction with DNA from the Fc-EMP fusion strain #3718 (see Example 3) using the sense.

primer 1216-52 and the antisense primer 2295-89 (SEQ ID NOS:.:369 and 1112, respectively). The nucleotides encoding the TNF-a inhibitory peptide were provided by the PCR primer 2295-89 shoiwnielow: 1216-52 AAC ATA AGT ACC TGT AGG ATC G 2295-89 CCG CGG ATC CAT TAC GGA CGG TGA CCC AGA GAG GTG TTT TTG TAG -115- TGC GGC AGG AAG TCA CCA CCA CCT CCA CCT TTA CCC The oligonucleotide 2295-89 overlaps the glycine linker and Fc portion of the template by 22 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested with restriction endonucleases Ndel and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. col strain 2596 cells as described for EMP-Fc hereini. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4544.

The nucleotide and amino acid sequences (SEQ ID NOS: 1055 and 1056) of the fusion protein are shown in Figures 19A and 19B.

TNF-a inhibitor-Fc. A DNA sequence coding for a TNF-a inhibitory peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. The template for the PCR reaction was a plasmid containing an unrelated peptide fused via a five glycine linker to Fc. The nucleotides encoding the TNF-a inhibitory peptide were provided by the sense PCR primer 2295-88, with primer 1200-54 serving as the antisense primer (SEQ ID NOS: 1117 and 407, respectively). The primer sequences are shown below: 2295-88 GAA TAA CAT ATG GAC TTC CTG CCG CAC TAC AAA AAC ACC TCT CTG GGT CAC CGT CCG GGT GGA GGC GGT GGG GAC AAA ACT 1200-54 GTT ATT GCT CAG CGG TGG CA The oligonucleotide 2295-88 overlaps the glycine linker and Fc portion of the template by 24 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame.

-116- The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4543.

The nucleotide and amino acid sequences (SEQ ID NOS: 1057 and 1058) of the fusion protein are shown in Figures 20A and 20B. Expression in E. coli. Cultures of each of the pAMG21-Fc-fusion constructs in E. coli GM221 were grown at 37 °C in Luria Broth medium containing 50 mg/ml kanamycin. Induction of gene product expression from the luxPR promoter was achieved following the addition of the synthetic autoinducer N-(3-oxohexanoyl)-DL-homoserine lactone to the culture media to a final concentration of 20 ng/ml. Cultures were incubated at 37 °C for a further 3 hours. After 3 hours, the bacterial cultures were examined by microscopy for the presence of inclusion bodies and were then collected by centrifugation. Refractile inclusion bodies were observed in induced cultures indicating that the Fc-fusions were most likely produced in the insoluble fraction in E. coli. Cell pellets were lysed directly by resuspension in Laemmli sample buffer containing 1-mercaptoethanol and were analyzed by SDS-PAGE. In each case, an intense coomassie-stained band of the appropriate molecular weight was observed on an SDS-PAGE gel.

Purification of Fc-peptide fusion proteins. Cells are broken in water (1/10) by high pressure homogenization (2 passes at 14,000 PSI) and inclusion bodies are harvested by centrifugation (4200 RPM in J-6B for 1 hour). Inclusion bodies are solubilized in 6M guanidine, 50mM Tris, 8mM DTT, pH 8.7 for 1 hour at a 1/10 ratio. The solubilized mixture is diluted 117 times into 2M urea, 50 mM tris, 160mM arginine, 3mM cysteine, pH The.mixture is stirred overnight in the cold and then concentrated about fold by ultafiltration. It is.then diluted 3 fold with 10mM Tris, urea, pH 9. The pH of this mixture is then adjusted to pH 5 with acetic acid. The precipitate is removed by centrifugation and the supernatant is loaded onto a SP-Sepharose Fast Flow column equilibrated in NaAc, 100 mM NaCI, pH 5 (10mg/ml protein load, room temperature).

The protein is eluted from the column using a 20 column volume gradient in the same buffer ranging from 100mM NaCI to 500mM NaCI. The pool from the-column is diluted 3 fold and loaded onto a SP-Sepharose HP column in 20mM NaAc, 150mM NaC, pH 5(10mg/ml protein load, room temperature). The protein is eluted using a 20 column volume gradient in the same buffer ranging from 150mM NaCI to 400mM NaCI. The peak is pooled and filtered.

Characterization of activity of Fc-TNF-a inhibitor and TNFinhibitor -Fc. Binding of these peptide fusion proteins to TNF- a can be characterized by BIAcore by methods available to one of ordinary skill in the art who is armed with the teachings of the present specification.

Example IL-1 Antagonists Fc-IL-1 antagonist. A DNA sequence coding for the Fc region of human IgGI fused in-frame to a monomer of an IL-1 antagonist peptide was constructed using standard PCR technology. The Fc and 5 glycine linker portion of the molecule was generated in a PCR reaction with DNA from the Fc-EMP fusion strain #3718 (see Example 3) using the sense primer 1216-52 and the antisense primer 2269-70 (SEQ ID NOS: 369. .and 1118, respectively). The nucleotdes encoding the IL-1-anagonist peptide were provided by the PCR primer 2269-70 shown below: -118- 1216-52 AAC ATA AGT ACC TGT AGG ATC G 2269-70 CCG CGG ATC CAT TAC AGC GGC AGA GCG TAC GGC TGC CAG TAA CCC GGG GTC CAT TCG AAA CCA CCA CCT CCA CCT TTA CCC The oligonucleotide 2269-70 overlaps the glycine linker and Fc.portion of the template by 22 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested with restriction endonucleases Ndel and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amrgen 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 antagonist-Fc. A DNA sequence coding for an IL-1 antagonist peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. The template for the PCR reaction was a plasmid containing an unrelated peptide fused via a five glycine linker to Fc. The nucleotides encoding the IL-1 antagonist peptide were provided by the sense PCR primer 2269-69, with primer 1200-54 serving as the antisense primer (SEQ ID NOS: 1119 and 407, respectively). The primer sequences are shown below: 2269-69 GAA TAA CAT ATG TTC GAA TGG ACC CCG GGT TAC TGG CAG CCG TAC GCT CTG CCG CTG GGT GGA GGC GGT GGG GAC AAA ACT 1200-54 GTT ATT GCT CAG CGG TGG CA -119- The oligonucleotide 2269-69 overlaps the glycine linker and Fc portion of the template by 24 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion o1 having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4505.

The nucleotide and amino acid sequences (SEQ ID NOS: 1061 and 1062) of the fusion protein are shown in Figures 22A and 22B. Expression and purification were carried out as in previous examples.

Characterization of Fc-IL-1 antagonist petide and IL-1 antagonist peptide-Fc activity. IL-1 Receptor Binding competition between IL-lp,

IL-

1RA and Fc-conjugated IL-1 peptide sequences was carried out using the IGEN system. Reactions contained 0.4 nM biotin-IL-1R 15 nM IL-1-TAG 3 uM competitor 20 ug/ml streptavidin-conjugate beads, where competitors were IL-1RA, Fc-IL-1 antagonist, IL-1 antagonist-Fc).

Competition was assayed over a range of competitor concentrations from 3 uM to 1.5 pM. The results are shown in Table C below: -120- Table C-Results from ML-1 Receptor Binding Competition Assay IL-lpep-Fc FO-L-lpep 59.58 112.2 IL-1ra 1.405 2.645

KI

281.5 530.0 Confidence Intervals EC50

KI

280.2 to 1002 148.9 to532.5 54.75 to 229.8 29.08 to 122.1 1.149 to 6.086 0.61061to 3.233 Goodness of Fit 0.9790 0.9790 ~0.9687 090 0.9602 121 Example-6 VEGE-Antagonis ts Fc-VEGF A ntagonist. A DNA sequence coding for the Fc region of human IgGI fused in.-franie to a monomer of the VEOF m-imetic peptide was constructed using- standArd PCR technology.. The templates for the PCR reaction were the pFc-A3 plasniid and a synthetic VIEGF midmetic peptide gene. The synthetic gene was assembled by annealing the' following two oligonucleotides primer (SEQ ID NO.S: 1110. and 1111, respectively): 2293-11 GTT GAA CCG AAC TGT GAC ATC CAT GTT ATG TGG GAA TGG GAA 2293 -12 CAG ACG TTC AAA ACA TTC CCA TTC CCA CAT AAC ATG CAT GTC ACA GTT CGG TTC AAC The two oligonucleotides anneal to form the following duplex encoding an amino acid sequence shown below (SEQ ID NOS 1113 and 1114): 57

CAACTTGGCTTGACACTGTAGGTACA.ATACACCCTTACCCTTACAALCTTGCA.AC

V E P N C D Z H VMXWE WE C F E R L a This duplex was amplified in a PCR reaction usin g 2293-05 a.nd 2293-06 as the sense and antisense primers (SEQ ID NOS. 1122 and 1123).

The Fc portion. of the molecule was generated in a PCR reaction with the pFc-A3 plasinid using the primers 2493-03 and 2293-04 as the sense and antisense primers (SEQ ID NOS. 1120 and 112 i,-resp-ectively).

The. full length fusion gene was obtained from a third PCR reaction using .the outside primers 2293-03 and 2293-06. These primers are shown beloW: 122 2293-03 ATT TGA TTC TAG AAG GAG GAA TAA CAT ATG GAC AAA ACT CAC ACA TGT 2293-04 GTC ACA GTT CGG TTC AAC ACC ACC ACC ACC ACC TTT ACC CGG AGA CAG GGA 2293-05 TCC CTG TCT CCG GGT AAA GGT GGT GGT GGT GGT GTT GAA CCG AAC TGT GAC ATC 2293-06 CCG CGG ATC CTC GAG.TTA CAG ACG TTC AAA ACA TTC CCA The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdeI and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4523.

The nuceotide and amino acid sequences (SEQ ID NOS: 1063 and 1064) of the fusion protein are shown in Figures 23A and 23B.

VEGF antagonist -Fc. A DNA sequence coding for a VEGF mimetic peptide fused in-frame to the Fc region of human IgG1 was constructed using standard PCR technology. The templates for the PCR reaction were the pFc-A3 plasmid and the synthetic VEGF mimetic peptide gene described above. The synthetic duplex was amplified in a PCR reaction using 2293-07 and 2293-08 as the sense and antisense primers (SEQ ID NOS. 112&4 and 1125 respectively).

The Fc portion of the molecule was generated in a PCR reaction with the pFc-A3 plasmid using the primers 2293-09 and 2293-10 as the sense and antisense primers (SEQ ID NOS. 1126 and .1127, respectively).

123 The full lengit fusion gene was obtained from a third PCR reaction using the outside primers 2293-07 and 2293-10. These primers are shown below: 2293-07 ATT TGA TTC TAG AAG GAG GAA TAA CAT ATG GTT GAA CCG AAC TGT GAC 2293-08 ACA TGT GTG AGT TTT GTC ACC ACC ACC ACC ACC CAG ACG TTC AAA ACA TTC 2293-09 GAA TGT TTT GAA CGT CTG GGT GGT GGT GGT GGT GAC AAA ACT CAC ACA TGT 2293-10 CCG CGG ATC CTC GAG TTA TTT ACC CGG AGA CAG GGA GAG The PCR gene product (the full length fusion gene) was digested with restriction endonucleases Ndel and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such done was selected and designated Amgen strain #4524.

The nucleotide and amino acid sequences (SEQ ID NOS: 1065 and 1066) of the fusion protein are shown in Figures 24A and 24B. Expression and purification were carried out as in previous examples.

Example 7 MMP Inhibitors Fc-MMF inhibitor. A DNA sequence coding for the Fc region of human IgGI fused in-frame to a monomer of an MMP inhibitory peptide was constructed using standard PCR technology. The Fc and 5 glycine linker portion of the molecule was generated in a PCR reaction with DNA from the Fc-TNF-a inhibitor fusion strain #4544 (see Example 4) using the sense primer 1216-52 and the antisense primer 2308-67 (SEQ ID NOS: 369 124 and 1115 ,respectively). The nucleotides encoding the MMP inhibitor peptide were provided by the PCR primer 2308-67 shown below: 1216-52 AAC ATA AGT ACC TGT AGG ATC G 2308-67 CCG CGG ATC CAT TAG CAC AGG GTG AAA CCC CAG TGG GTG GTG CAA CCA CCA CCT CCA CCT TTA CCC The oligonudeotide 2308-67 overlaps the glycine linker and Fc portion of the template by 22 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested with restriction endonucleases Ndel and BamHI, and then ligated into the vector.pAMG21 and transformed into competent E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nudeotide sequence. A single such done was selected and designated Amgen strain #4597.

The nucleotide and amino acid sequences (SEQ ID NOS: 1067 and 1068) of the fusion protein are shown in Figures 25A and 25B. Expression and purification were carried out as in previous examples.

MMP Inhibitor-Fc. A DNA sequence coding for an MMP inhibitory peptide fused in-frame to the Fc region of human IgGI was constructed using standard PCR technology. The Fc and 5 glycine linker portion of the molecule was generated in a PCR reaction with.DNA from the Fc-TNF-a inhibitor fusion strain #4543 (see Example The nuceotides encoding the MMP inhibitory peptide were provided by the sense PCR primer 2308- 66, with primer 1200-54 serving as the antisense primer (SEQ ID NOS: 1116 and 407,-respectively). The primer sequences are shown below: 2308-66 GAA TAA CAT ATG TGC ACC ACC CAC TGG GGT TTC ACC CTG TGC GGT. GGA GGC. GT GGG GAC AAA 1200-54 GTT ATT GCT CAG CGG TGG CA -125- .The oligonucleotide 2269-69 overlaps the glycine linker and Fc portion of the template by 24 nucleotides, with the PCR resulting in the two genes being fused together in the correct reading frame.

The PCR gene product (the full length fusion gene) was digested with restriction endonucleases NdI and BamHI, and then ligated into the vector pAMG21 and transformed into competent E. coli strain 2596 cells as described for EMP-Fc herein. Clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence. A single such clone was selected and designated Amgen strain #4598.

The nucleotide and amino acid sequences (SEQ ID NOS: 1069 and 1070) of the fusion protein are shown in Figures 26A and 26B.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto, without departing from the spirit and scope of the invention as set forth herein.

Abbreviations 0Abbreviations used throughout this specification are as defined below, unless otherwise defined in specific circumstances.

Ac acetyl (used to refer to acetylated residues) AcBpa acetylated p-benzony-L-phenylalanine ADCC antibody-dependent cellular cytotoxicity Aib aminoisobutyric acid bA beta-alanine Bpa p-benzoyl-L-phenylalanine BrAc bromoacetyl (BrCH2

C(O)

-126-

BSA

Bzl Cap

CTL

CTLA4

DARC

DCC

Dde

EMP

ESI-MS

EPO

Fmoc.

G-CSF

,GH

HCT

HGB

hG.H HOBt

HPLC

IL

IL-R

IL-1R IL-Ira.

Lau

LPS

LYMPH

MALDI-S

Bovine serum albumin Benzyl Caproic acid Cytotoxic T lymphocytes Cytotoxic T lymphocyte antigen 4 Duffy blood group antigen receptor Dicylcohexylcarbodiimide 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyL Erythropoietin-mimetic peptide Electron spray ionization mass spectrometry Erythropoietin fluorenylmethoxycarbonyl Granulocyte colony stimulating factor Growth hormone henatocrit hemoglobin Human growth hormone I-Hydroxybenzotriazole high performance liquid chromatography interleukin interleukin receptor interleukin-l receptor interleukin-l receptor antagonist Lauric acid lipopolysaccharide lymphocytes Matrix-assisted laser desorption ionization-mass spectrometry methyl -127- MeO-

MHC

MMP

MMPI

1-Nap

NEUT

NGF

Me

NMP

PAGE

PBS

Pbf

PCR

Pec

PEG

pGlu Pic

PLT

pY

RBC

RBS

RT

Sar

SDS

STK

t-Boc tBu

TGF

THF

methoxy major histocompatibility complex matrix metalloproteinase matrix metalloproteinase inhibitor 1-napthylalanine neutrophils nerve growth factor norleucmne N-methyl-2-pyrrolidinone polyacrylanide gel electrophoresis Phosphate-buffered saline 2,2,4,6,7-pendamethyldihydrobenzofural-5-sufoflyI polymerase chain reaction pipecolic acid Poly(ethylene glycol) pyroglutamic acid picolinic acid platelets phosphotyrosine red blood cells ribosome binding site room temperature (25 0 sarcosine sodium dodecyl sulfate serine-threonine kinases tert-Butoxycarbonyl tert-ButylI tissue growth factor thymic humoral factor -12 8- TK tyrosine kinase TMP Thrombopoietin-mimetic peptide TNF Tissue necrosis factor TPO Thrombopoietin.

TRAIL TNF-related apoptosis-inducing ligand, Trt trityl UK urokinase UKR urokinase receptor VEGF vascular endothelial. cell growth factor( VIP vasoactive intestinal peptide WBC white blood cells

Claims (9)

1. A composition of matter of the formula and multimers thereof, wherein: F' is an Fc domain; X' and X 2 are each independently selected from -P 2 2 3 and P1, and P' are each independently randomized GCSF binding peptide sequences; L 2 L 5 and L' are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1 and wherein "peptide" refers to molecules 2 to 40 amino acids and wherein neither X' nor X 2 is a native protein.

2. The composition of matter of Claim 1 of the formulae X'-F' or F'-X 2

3. The composition of matter of Claim 1 of the formula

4. The composition of matter of Claim 1 of the formula 2 The composition of matter of Claim 1 wherein F' is an IgG Fc domain.

6. The composition of matter of Claim 1 wherein F' is an IgG1 Fc domain,

7. The composition of matter of Claim 1 wherein F' comprises the sequence of SEQ ID NO: 2.

8. A DNA encoding a composition of matter of Claim 1.

9. An expression vector comprising the DNA of Claim 8. r

130- A host cell comprising the expression vector of Claim 9. 11. The cell of Claim 10, wherein the cell is an E. coli cell. 12. A process for preparing a GCSF binding compound, which comprises a) selecting at least one randomized non-native GCSF-binding peptide of 2 to 40 amino acids in length; and b) preparing a GCSF binding compound comprising at least one Fc domain covalently linked to at least one amino acid sequence of 0the selected peptide or peptides. 13. The process of Claim 12, wherein the peptide is selected in a process comprising screening of a phage display library, an E. coli display library, a ribosomal library, or a chemical peptide library. 14. The process of Claim 12, wherein the Fc domain is an IgG Fc domain. The process of Claim 12, wherein the Fc domain is an IgG1 Fc domain. 16. The process of Claim 12, wherein the Fc domain comprises the sequence of SEQ ID NO: 2. 17. The process of Claim 12, wherein the compound prepared is of the formula 2 )b and multimers thereof, wherein: F 1 is an Fc domain; X 1 and X 2 are each independently selected from -(L 1 )c-P 1 (L 2 )d _P2, -(L 1 c -P -(L 2 )dP 2 -(L 3 )e-P 3 and 2 )d-P2-(L 3 )e -P3-(L 4 )f-P 4 P2, P3, and P4 are each independently sequences of pharmacologically active peptides; L 2 L 3 and L 4 are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1, provided that at least one of a and b is 1. 131 18. The process of Claim 17, wherein the compound prepared is of the formulae X'-F 1 or F-X 2 19. The process of Claim 17, wherein the compound prepared is of the formulae FI-(L1)c-P1 or F 1 -(L 1 c -P 1 -(L 2 )d-P 2 The process of Claim 17, wherein F 1 is an IgG Fc domain. 21. The process of Claim 17, wherein F 1 is an IgG1 Fc domain. 22. The process of Claim 17, wherein F 1 comprises the sequence of SEQ ID NO: 2. 23. A composition of matter of the formula (Xl)a-Fl-(X 2 )b and multimers thereof, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 24. A DNA encoding a composition of matter, an expression vector and a host cell, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. A process for preparing a GCSF binding compound, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.