AU2004203690B2

AU2004203690B2 - Agonist peptide dimers

Info

Publication number: AU2004203690B2
Application number: AU2004203690A
Authority: AU
Inventors: Dana L Johnson; Robert Allan Zivin
Original assignee: Ortho McNeil Pharmaceutical Inc
Current assignee: Janssen Pharmaceuticals Inc
Priority date: 1995-06-07
Filing date: 2004-08-09
Publication date: 2008-02-14
Anticipated expiration: 2016-06-06
Also published as: AU5433701A; AU2004203690A1

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant/s: Ortho-McNeil Pharmaceutical, Inc.

Actual Inventor/s: Dana L Johnson and Robert Allan Zivin Address for Service is: SHELSTON IP Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: AGONIST PEPTIDE DIMERS Details of Original Application No. 54337/01 dated 12 Jul 2001 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 30685AUP01 500407857 1.DOC/5844 -la- AGONIST PEPTIDE DIMERS FIELD OF THE INVENTION The present invention relates to the dimerization of agonists and antagonists of cell surface receptors and particularly to peptide dimers which behave as cell surface receptor agonists in their dimeric form. Such receptors belong to the dimerization-mediated activation class often observed among receptors for growth and differentiation factors. The agonists of this class of receptors is understood to effect dimerization of the receptor and thus signal initiation. The present invention exemplifies dimers of erythropoietin (EPO) agonists and antagonists comprising a core amino acid sequence of X 3

X

4

X

5

GPX

6

TWX

7

X

8 (SEQ ID NO: 1) wherein each amino acid is indicated by standard one letter abbreviation; X 3 can be C, A, c-amino-y-bromobutyric acid or Hoc; X 4 can be R, H, L or W; X 5 can be M, F, or I; X 6 is independently selected from any one of the 20 genetically coded L-amino acids or the stereoisomeric D-amino acids; X7 can be D, E, I, L or V; and X 8 can be C, A, a-amino-y-bromobutyric acid or Hoc, provided that either X 3 or X 8 is C or Hoc.

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.

Erythropoietin (EPO) is a glycoprotein hormone with an approximate molecular weight of 34,000 daltons. The primary role of EPO, which is synthesized in the kidneys of mammals, is to stimulate mitotic cell division and differentiation of erythrocyte precursor cells. As a result, EPO acts to stimulate and to -2- 1 regulate the production of erythrocytes. Erythrocytes, and the hemoglobin contained therein, play a central.

role in supplying oxygen to the body. Thus, the stimulation of erythrocyte production is able to increase the oxygen-carrying capacity of the blood.- During normal conditions, EPO is present in very low concentrations in plasma. Under hypoxic conditions, the amount of EPO in the circulation is increased in response to reduced.0, blood levels.

Hypoxia may be caused from various conditions including the loss of large amounts of blood, destruction of red blood cells by over-exposure to radiation or chemotherapeutic agents, reduction in oxygen intake due Sto high altitudes or prolonged unconsciousness, or by various forms of anemia. As the hypoxic condition diminishes, the amount of EPO produced subsequently diminishes.

Because of the essential role of EPO in red blood cell formation, the hormone is useful in both the diagnosis and the treatment of blood disorders characterized by low or defective red bl6od cell production. Recent studies provide a basis for the efficacy of EPO therapy in a variety of disease states, disorders, and states of hematologic irregularity, including: beta-thalassemia (See, Vedovato et al.

(1984) Acta. Haematol. 71:211-213); cystic fibrosis (See, Vichinsky et al. (1984) J. Pediatric 105:15-21); pregnancy and menstrual disorders (See, Cotes et al.

(1983) Brit. J. Ostet. Gyneacol. 90:304-311); early 3 anemia of prematurity (See, Haga et al. (1983) .c-a Pediatr. Scand. 72:827-831); spinal cord injury (See, -3- Claus-Walker et al. (1984) Arch. Phvs. Med. Rehabil.

65:370-374); space flight (See, Dunn.et al. (1984) Eur.

J. ADD1. Phvsiol. 52:178-182); acute blood loss (See.

Miller et al. (1982) Brit. J. Haematol. 52:545-590); aging (See, Udupa et al. (1984) J. Lab. Clin. Med.

103:574-588); various neoplastic disease states accompanied by abnormal erythropoiesis (See, Dainiak et al. (1983) Cancer 5:1101-1106); and renal insufficiency (See, Eschbach et al. (1987) N. Ena. J. Med. 316:73-78).

Although purified, homogenous EPO has beencharacterized, little is known about the mechanism of EPO-induced erythroblast proliferation and differentiation. The specific .interaction of EPO with progenitor cells of immature red blood cells, platelets, and megakaryocytes has not been described. This is due in part, to the small number of surface EPO receptor molecules on normal erythroblasts and on the erythroleukemia cell lines. See Krantz and Goldwasser (1984) Proc. Natl. Acad. Sci. USA, 81:7574-7578; Branch et al. (1987) Blood 69:1782-1785; Mayeux et al. (1987) FEES Letters 211:229-223; Mufson and Gesner (1987) Blood 69:1485-1490; Sakaguchi et al. (1987) Biochem. Biophvs.

Res. Commun. 146:7-12; Sawyer et al. (1987) Proc. Natl.

Acad. Sci. USA 84:3690-3694;. Sawyer et al. (1987) J.

Biol. Chem. 262:5554-5562; and Todokoro et al. (1988) Proc. Natl. Acad. Sci. USA 84:4126-4130. The DNA sequences and encoded peptide sequences for murine and human EPO receptor proteins have been described. See, D'Andrea et al. PCT Patent Publication No. WO 90/08822 published 1990).

(published 1990).

-4- The EPO-recerutzr (EPO-R) belongs to the CL-Ls of 'growth-factor-type receptoars which axe activated by a 1igand-induced protein dimerization. Other hormcnes and cYtokines such as human growth hox-one .g=inulocyte colony stimulating factbr epidea I growth factor (EGF) and insuli~n can cross-link twio Xecepor resulting i.juxtapositilon of two cytoplasmic tails. M(any of these dimerization- activated receptors have prote in k-4 nase, domains= within the cytoplasmic tails l0 that. phosphoylate: the* neighboring tail upon d4imeization While some cyrtoplasmic ta-ils lack intrinsic kinase- activity, these function by as sociation with protein. kinases. The EPO receptor is of the latiter type. In each ca, pkosphorylation results in -the activatidn' of a signalijng pathway.

In. accordance with, the present inetoit has been discovered that the dimerization of'peptiLde agoists an~d antagonists of dimerization-mediazed' *receptors, such as. EPO-R, increase the biological efficacy. relative to -the biologi cal' activity of the 'monomeic'. agonizzas and al'ters. the~ properties of the antagonists such that-, these dimez function a~s agonists-, exh Ibiting -biological actlvity-, SUMMVALRY OF THE DiVENTION The present invention relates to peptide dimners which behave as cell-surface receptor agonists, dimers which exhibit binding and signal initiation of growth factor-type receptors. The present invention further relates to peptide dimers which behave as EPO agonists. These dimers have two 'monomeric' peptide units of 10 to 40 or more emino acids,. preferably 14 to about 20 residues in length, comprising a core amino acid sequence of

X

3 X,XGPXTWX,X, (SEQ ID NO: 1) where each amino acid is indicated by standard one letter abbreviation; X, can be C, A, a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine; X 4 can be R, H, L, or W; X, can be M, F, or I; X, is independently selected from any one of the genetically coded L-amino acids or the stereoisomeric D-amino acids; X, can be D, E, I, L, or V; and X. can be C, A, a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine, provided that either X, or X, is C or Hoc.

Preferably, the monomeric peptide unit of the dimer .comprises a core sequence YXX,XX.XGPXTWX,X, (SEQ ID NO: 2) where each amino acid is indicated by standard one letter abbreviation; each X, and X, is independently selected from any one of the 20 genetically coded Lamino acids; X3 can be C, A, a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine; X. can be R, H, L, or W; X, can be M, F, or I; X, can be D, E, I, L, or V; and X. can be C, A, a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine, provided that either X 3 or X. is C or Hoc.

More preferably, the monomeric peptide unit of 25 the dimer comprises a core sequence of amino acids

XIYXX

3 XXsGPXTWXXeXXoXII (SEQ ID NO: where each amino acid is indicated by standard one letter abbreviation; each X1, X 2 X1o, and is independently selected from any one of the genetically coded L-amino acids; X, can be C, A, aamino-y-bromobutyric acid, or Hoc, where Hoc is hoinocysteine; X 4 can be R, H, L, or W; X 5 can be M, F, or I; X 7 can be D, E, I, L or V; and X 8 can be'C, A, a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine, provided that either X 3 or X 8 is C or Hoc.

Further, the present invention relates to a 4Aimer -in which both X 3 and X 8 are C and thus, the monomeric peptide unit of the dimer comprises a core. sequence of amino acids X.yxcx.XSGpxEsTwx.cxx 0 xJ.- (SEQ ID NO: more preferab ly, the monomeric peptide unit Comprises a core sequence of -amino acids XL.yx 2 cZx 4 5 GpX.,TWX 7

,CX.XICX

2

(SEQ

ID NO: 5),1 where X, can be R or H; Xe can be F or lo can be 1, L, T, M, or V; is D or V; X9 can be G, K, L, Q# S, -or and X3.

0 can be A, P, R, or.Y. In a most preferred embodiment, the monomeric peptide unit of the dimer will comprise' a core sequence of amino acids X:fLY 2 CXXsGPXECX.X Lx, 2 (SEQ ID NO: where X 3 can be D, E r L, N, S, T, or. V; X 2 can be S, K, L, M, S, or T; X*is RorH; X9 cartbe K, R, S, or T; and J P. Particularly jpreferd monomeric peptide units of the. dimers include:

QMLCFGPVTWDCQKG

GGTYSCHFGPLTWVMCPQGG

GGDYCEMGPLTWVCKPLGG

VG1NYM GPITWVCRPGGG GGVYAdMNWITWVCSPLGG

VGNYMAHKGPITWVCRPGG

GLYAcMMGPMTWvcQPLRG

TIAQYICMGPETWECPSPPJ

YScZFGPLTWVCK Y UFGPLTWVC (SEQ MD (SEQ ID (SEQ ID (SEQ MD (SEQ ID (SEQ ID (SEQ MD (SEQ ID (SEQ ID (SEQ, ID (SEQ ID

NO:

7); 12); 13); 14); is); 17); and SCHFGPLTWVCK (SEQ ID NO: 18).

Other monomeric peptide units of the present dimers include peptides comprising a core sequence of the formula (AX 2 nX 3

X

4

X

5

GPX

6

TWX

7 X (SEQ ID NO: 19) wherein

X

2 through X8 are as previously defined herein (SEQ ID NO: n is 1 or 0 and A is any one of the naturally occurring L-amino acids except Y (tyrosine); n is defined herein as the number of occurrences of(AX 2 which can be 1 or none in the core sequence. When

(AX

2 is present, i.e. when n 1, A is not tyrosine and A is not any non-naturally occurring aromatic amino acid analog. Such monomeric peptide units of the dimers of this invention can be prepared by truncating the peptides of Fig. 9, for example, from the N-terminus to delete the Y, tyrosine residue in SEQ ID NOS. 21-93. Such monomeric peptides can also be prepared by substitution of Y in position A in the peptides of Fig. 9.

In accordance with the present invention the monomeric units of the dimers can be the same or different.

In a related invention, polyethylene glycol (PEG) is employed as a linker to form the dimeric peptides described herein through a covalent bond.

The present invention is further related to pharmaceutical compositions comprising at least one dimer peptide described herein and a pharmaceutical carrier.

The present invention relates to a method for therapeutically treating a mammal having a condition resulting from a hormone or growth factor deficiency by administration of at least one of the dimer peptides of the present invention.

The present invention also relates to a method for therapeutically treating a mammal having a condition resulting from a deficiency of EPO or from reduced levels of blood oxygen caused by a decrease in erythrocyte number.

Nl This invention also relates to a method for preparing agonists of cell-surface c' receptors wherein agonists of the class of cell-surface or dimerization-mediated receptors are dimerized to enhance the in vitro or in vivo biological activity of the cell-surface receptor relative to the monomeric agonist from which the dimer is derived.

The method is also directed to the preparation of agonists of such growth-factor-type receptors by dimerizing antagonists of these receptors; the dimerized 'antagonists' thereby exhibit agonist biological activity in vitro and in vivo. The present method also relates to the preparation of EPO-R dimer agonists from monomeric EPO-R antagonists.

According to a first aspect, the present invention provides a method of improving the bioactivity of a cell surface receptor comprising dimerizing a monomeric agonist of said cell surface receptor to form a dimer wherein each said monomeric agonist is a peptide of between 10 to 40 amino acids and contacting said dimer with said cell surface receptor to effect said improved biological activity wherein the agonist of said cell surface receptor is a GH agonist, PDGF agonist, EGF agonist, G-CSF agonist, TPO agonist, VEGF agonist, FGF agonist, insulin agonist, IL-3 agonist, IL-5 agonist, IL-6 agonist or IL-2 agonist.

8a- According to a second aspect, the present invention provides a method of activating a cell surface receptor to induce biological activity of said cell surface receptor comprising dimerizing a monomeric agonist of said cell surface receptor to form a dimer wherein each said monomeric agonist is a peptide of between 10 to amino acids and contacting said formed dimer with said receptor thereby inducing said biological activity wherein the agonist of said cell surface receptor is a GH agonist, PDGF agonist, EGF agonist, G-CSF agonist, TPO agonist, VEGF agonist, FGF agonist, insulin agonist, IL-3 agonist, IL-5 agonist, IL-6 agonist or IL-2 agonist.

According to a third aspect, the present invention provides a method of preparing a cell surface receptor agonist comprising dimerizing a cell surface receptor antagonist to form a dimer each said antagonist being a peptide of between 10 to 40 amino acids wherein said cell surface antagonist is a GH antagonist, PDGF antagonist, EGF antagonist, G-CSF antagonist, EGF antagonist, GM-CSF antagonist, TPO antagonist, VEGF antagonist, FGF antagonist, FGF antagonist, insulin antagonist, IL-3 antagonist, IL-5 antagonist, IL-6 antagonist, or an IL-2 antagonist.

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".

BRIEF DESCRIPTION OF THE DRAWINGS Fig. I shows a major peak, with a retention time of 37 minutes, of the dimerized EPO peptide, GGTYSCHFGPLTWVCKPQGG (SEQ ID NO:8) 1 Fig. 2 shows a major peak, with a retention time of 48 minutes, following purification of the dimerized EPO peptide, (SEQ ID NO: 8).

Fig. 3 depicts the MALDI-TOF mass spectral analysis of the dimerized peptides, including peptide (SEQ ID NO: GGTYSCHFGPLTWVCKPQ (SEQ ID NO: 13) and SCHFGPLTWVCK (SEQ ID NO: 18).

Fig. 4 shows the SDS-PAGE analysis of DPDPB crosslinking of EPO binding protein (EBP) in the presence and absence of EPO agonist peptides.

Fig. 5 demonstrates equilibrium EPO binding to immobilized EPO binding protein. Panel A represents the equilibrium binding data and Panel B (inset) is the linear transformation (Scatchard) of the data set in S Panel

A.

Fig. 6 depicts the results of a competitive binding assay run on-the EPO agonist peptide (SEQ ID NO: 8) in competitive binding with '-I]EPO to EBP beads (Panel and EPO responsive cell 2 proliferation studies in FDC-P1 derived cell lines containing either a human (Panel B) or murine EPO receptor (Panel C).

Fig. 7 is a graphic representation of the results of the exhypoxic mouse bioassay; stimulation of the incorporation of 9 Fe] into nascent red blood cells by EPO, peptide (SEQ ID NO: 8) (Panel A) and peptide (SEQ ID NO: 8) dimer (Panel B).

Fig. 8 demonstrates the effect of PEG dimerization of peptide (SEQ ID NO: 18) activity in EPO 30 responsive cell proliferation studies in FDC-F1 derived cell lines containing a human EPO receptor.

1 Fig. 9 provides the sequences of reprE.sentative monomeric peptides of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to peptide dimers which behave as cell surface receptor agonists, dimers which exhibit binding and signal initiation of growth-factor-type receptors. Sometimes called cellsurface receptors, growth-factor-type receptors or dimerization-mediated activator-receptors, these are a class of molecules which are understood to be activated by ligand-induced or ligand stabilized dimerization.

Agonists of such receptors typically include large polypeptide hormones including the cytokines, insulin and various other growth or differentiation factors.

The agonists are understood to induce dimerization of the receptor and thereby effect signal initiation. Such agonists are believed to effectively cross-link two receptors resulting in the repositioning of cytoplasmic tails which may directly or indirectly effect phosphorylation of the cytoplasmic tails and activation of a signaling pathway.

The present invention specifically includes those molecules which behave as agonists of cell-surface receptors when dimerized in accordance with this invention. Such dimer agonists can include 'monomeric' units which exhibit agonist or antagonist activity for the related receptor molecule and may be the same or different. The dimers are preferably peptides but may alternatively be small molecule pharmacophores. These molecules when dimerized exhibit agonist activity of -11cell-surface receptors 'in vitro and in vivo. Such receptors include, for example, EPO, GM-CSF, G-CSF, M- CSF, GH, EGF, PDGF, VEGF, Insulin and FGF. Other receptors which are activated by heterodimerization or multimerization may also be subject to activation by this mechanism including, IL-3, IL-5, IL-6, IL-2 and TPO. The dimers of the present invention have two 'monomeric' peptide units of 10 to 40 or more amino acids, preferably 14 to about 20 amino acid residues in length. In a preferred embodiment, these monomeric peptide units comprise a core sequence of amino acids X,X.X,GPX,TWX,Xe (SEQ ID NO: 1) where each amino acid is indicated by standard one letter abbreviation; X, can be C, A, a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine; X, can be R, H, L, or W; X, can be M, F, or I; X 6 is independently selected from any one of the genetically coded L-amino acids or the stereoisomeric D-amino acids; X, can be D, E, I, L, or V; and X, can be a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine, provided that either X, or Xe is C or Hoc.

Preferably, the monomeric peptide unit of the dimer comprises a core sequence YX 2

XX

4 XGPXTWXXe (SEQ ID NO: 2) where each amino acid is indicated by standard one letter abbreviation; each X, and X. is independently selected from any one of the 20 genetically coded .Lamino acids; X, can be C, A, a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine; X 4 can be R, H, L, or W; X, can be M, F, or I; X, can be D, E, I, L, or V; and Xe can be C, A, a-amino-y-bromobutyric acid, or Hoc, where Hoc is homocysteine, provided tha nirher X3 or Xe is C or Hoc.

-12- More preferably, the monomeric peptide unit of the dimer comprises ac,:re seauence of amino acids XlY~X 2

X

3 xXXGPXITWX,.XX,LX,. (SEQ ID. NO: where each amino acid is indicated by standard one letter abbreviation; each X 2 1 XG, X 9 and is independently selected-from any one of the genetically coded L-amino acids; X, can be C, A,.

a- amino bromobutyri c acid, or Hoc, where Hoc is homocysteine; X, can be R, H, L, or W; X. can be M, F, or I; can be D, E, 1, L or V; and X. can be C, A, a-amino-l-bromobutyric acid, or Hoc, where Hoc is homocysteine, provided that either X, or X, is C or Hoc.

In a more pref erred embodiment, both X, and X, will be C and thus, the monomeric peptide unit of the dimer comprises a core sequence of .amino acids XCYX XGPXTWXCXXOX,, (SEQ ID NO: More preferably, the mono. meric peptide unit comprises a core sequence of amino acids XYXXspx 4 TwX,cx 9

(SEQ

ID NO; where can be R or H; X. can be F or X 6 can be I, L, T, M, or V; X, is D or V; X. can be G, K,* L, Q, R, S, or T; and can be A, G, P, R, or Y. In a most preferred embodiment, the monomeric peptide-unit of the dimer comprises a core sequence of amino acids x Im. CX..XGPXTX,CX,X,,X,, (SEQ ID NO: where-X 1 can be D, E, L, S, T, or V; X, can be.A, H, K, L, M, S, or T; X, is R or H; X, can be K, R, S, or T; and X 1 is P. Particularly preferred monomeric peptide units of the present dimers include: GGLYLCRFGPVTWDCGYKGG .(SEQ ID NO: 7,; GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 8); -13- 1 GGDYHCRMGPLTWVCKPLGG (SEQ ID NO: 9); VGNYMCHFGPITWVCRPGGG (SEQ ID NO: GGVYACRMGPITWVCSPLGG (SEQ ID NO: 11); VGNYMAHMGPITWVCRPGG (SEQ ID NO: 12); GGTYSCHFGPLTWVCKPQ (SEQ ID NO: 13); GGLYACHMGPMTWVCQPLRG (SEQ ID NO: 14); TIAQYICYMGPETWECRPSPKA (SEQ ID NO: YSCHFGPLTWVCK (SEQ ID NO: 16); YCHFGPLTWVC (SEQ ID NO: 17); and SCHFGPLTWVCK (SEQ ID NO: 18).

The dimer peptides of the.present invention exhibit increased biological potency..in vitro and in vivo relative to the monomeric agonists from which the 5 dimers are derived. Moreover, cell surface receptor antagonists can be 'converted' to cell surface receptor agonists in accordance with the present invention.

Specifically, a cell surface receptor antagonist can be dimerized with PEG or another appropriate linker which permits mutual binding of the monomeric moieties with the receptors. As a result, the dimer exhibits effective binding to the target receptor and behaves as an agonist. Accordingly, the dimers of this invention demonstrate enhanced biological potency in vitro and in 25 vivo relative to their monomeric forms.

The dimer peptides of the present invention bind to and biologically activate the cell surface receptor or otherwise behave as agonists and are preferably formed by employing polyethylene glycol as a linker between the monomeric peptide units descr-bed herein. While other conventional chemical systems can -14- 1 also be employed to form the dimer peptides of this invention including using other known polymeric compounds, pegylation is preferred.

The linking compounds of the present invention include any molecule which covalently binds" the monomeric peptides at an appropriate distance or which otherwise effects dimerization of the particular cell surface receptor thereby initiating biological efficacy.

Starting with an appropriate synthetic peptide, containing a free amino group or other reactive site such as hydroxyls, carboxylic acids or sulfhydryls, the peptide is added in excess to a reaction mixture containing a corresponding reactive polymer. The polymer can be of a repeating nature such as polyethylene glycol, peptides, modified peptides or peptide analogs. Alternatively, the peptide can be dimerized on a small molecule scaffold such as activated benodiazepins, oxazolones, azalactones, aminimides or diketopiperazines. The most readily avail.bise linker of variable distance are ones based on linear unbranched polyethylene glycols.

The following is a schematic of a preferred preparatory methodology employing PEG succinimidyl propionate as the linker between the monomer units of the dimer peptides.

Scbgme 1.- 0 -a-i 2

CH

2 0--PEG (AD)-O-a4H-CHZC-' PEG scimidyl 1 xrprmte, (SPA)Z-?EG

H

2 4--R(MWJ61233) R2 equfvmeftS (RWJ 61233) R-N--C-CH 2 CHj 0--PEG ()CC 2 H C-N-RWJ 61233) A~MW: ca 7300 [2 aquivaeMQ~ Dimerization and especially pegylation in a head-to-head (amino to amino terminus) or head-to-tail (amino to carboxyl terminus) .configuration is preferred relative to internal covalent binding of the monomeric peptides. The 'monomer' units of the dimer peptides of the present invention can be the same or different, although the same are preferred.

The monomeric peptides which are used to form the dimers of the present invention can be prepared by classical chemical methods well known in the art. The standard methods include, for example, exclusive solid phase synthesis and recombinant DNA technology. See, Merrifield (1963) J. Am. Chem. Soc. 85:2149. Solid phase synthesis is typically commenced from the Cterminal end of the peptide using an a-emino protected resin. A suitable starting material can be prepared by attaching the required amino acid to a chloromethylated resin (such as BIO-BEADS SX-1, Bio Rad Laboratories, Richmond, CA), a hydrozymethyl resin, (described by Bodonszky et al. (1966) Chem. Ind.

-16- 1 (London) 38:1597) or a benzhydrylamine resin (described by Pietta and Marshall (1970) Chem. Commn. 650).

The a-amino protecting groups are those known to be useful in the art of stepwise synthesis or peptides. Included are acyl type protecting groups formyl, trifluoroacetyl, acetyl), aromatic urethane type protecting groups benzyloxycarboyl (Cbz) and substituted Cbz), aliphatic urethane protecting groups t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protecting groups benzyl and triphenylmethyl). The preferred.X-amino protecting group is Fmoc. The side chain protecting group (typically ethers, esters, trityl, PMC, and the like) remains intact during coupling and is not split off during the deprotection of the amino-terminus protecting group -or during coupling. The side chain protecting group must be removable upon the completion of the synthesis of the final peptide and under reaction conditions that will not alter the target peptide.

The side chain protecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. The side chain protecting groups for Asp include benzyl, 2,6dichlorobenzyl, methyl, ethyl, and cyclohexyl. The side chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6dichlorobenzyl, and Cbz. The side chain protecting groups for Thr and Ser are benzyl. The side chain protecting groups for Arg include nitro, Tosy. (Tcs), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts), or -17- 1 Boc. The side chain protecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2bromobenzyloxycarbonyl (2-BrCbz), Tos, or Boc.

After removal of the a-amino protecting group, the remaining protected amino acids are coupled stepwise in the desired order. Each protected amino acid is generally reacted in about a 3-fold excess using an appropriate carboxyl group activator such as 2-(1Hbenxotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU) or dicyclohexylcarbodiimide (DCC) in solution of methylene chloride or dimethyl formamide (DMF) mixtures.

After the desired amino acid sequence has been completed, the desired peptide is decoupled from the resin support by treating the mixture with a reagent such as trifluoroacetic acid (TFA) or hydrogen fluoride These reagents not only cleave the peptide from the resin, but also cleave all remaining side chain protecting groups. When the chloromethylated resin is used, hydrogen fluoride treatment results in the formation of the free peptide acids. When the benzhydrylamine resin is used, hydrogen fluoride treatment results directly in the free peptide amide.

Alternatively, when the chloromethylated resin is employed, the side chain protected peptide can be decoupled by treatment of the peptide resin with ammonia to give the desired side chain protected amide or with an alkylamine to give a side chain protected alkylamide or dialkylamide. Side.chain protection is then removed 30 in the usual fashion by treatment with hydrogen fluoride to give the free amides, alkylamides, or dialkylamides..

-18- 1 These prccdures can also be used to synthesize peptider in which amino acids other than the naturally occurring, genetically encoded amino-acids are substituted at one, two or more positions of any of the compounds of the invention. For instance, naphthylalanine can be substituted for tryptophan, facilitating synthesis. Other synthetic amino acids that can be substituted into the peptides of the present invention include L-hydroypropyl, L-3, 4- .amino acids such as L-6hydroxylysyl and D-6-methylalanyl, L-a-methylalanyl,

B

amino acids, and isoquinolyl. D-amino acids and nonnaturally occurring synthetic amino acids can also be incorporated into the peptides of the present invention.

In another embodiment -of the present invention, a method of enhancing the in vitro or in vivo biological potency of a cell surface receptor agonist is provided. This methodology is achieved by dimerizing the receptor, agonist with a linker molecule, such as PEG, to form an appropriate spatial relationship between the monomeric peptide units of the dimer and thereby permitting each of the constituents of the dimers to bind to their receptors to achieve enhanced biological potency, to dimerize and thereby activate the receptors to induce the relevant biological activity of the-parti-cular cell-surface receptor, e.g. EPO-R.

Biological activity can be measured by the skilled artisan in various in vitro and in vivo assays and as demonstrated in the examples of the present invention.

The peptide or molecule with binding affinity for a given receptor will have increased conformational -19flexibility leading to fewer barriers to effective receptor interaction and subsequently receptor activation. This is also indicated for molecules which can bind but not activate a receptor subtype in that such molecules can become more effective inhibitors of ligand binding.

The present invention further provides a method for altering a cell-surface receptor antagonist, S a molecule exhibiting receptor binding but no biological activity, to behave as a cell-surface receptor agonist in vitro or in vivo. This method is achieved by dimerizing the antagonist molecule with an appropriate linker molecule such as PEG, other polymerized molecules or a peptide. In a preferred embodiment, an EPO antagonist, i.e. a peptide exhibiting receptor binding but no biological EPO activity can be altered by dimerization to obtain a dimer which behaves as an EPO receptor agqnist. Thus, for example, in the case of EP3-R these include the monomeric peptide units of the present dimers comprising a core sequence of general formula (AX) ,XXXGPXTWXX. (SEQ ID NO: 19) wherein X, through X. are as previously defined herein, in (SEQ ID NO: n is 1 or 0 and.A..is any one of the naturally occurring L-amino acids except Y (tyrosine); n is.

defined herein as the number of occurrences of which can be 1 or none in the core sequence. When X, is present, when n 1, K is not tyrosine and A is not any non-naturally occurring aromatic amino acid analog. Such monomeric peptide units of the dimers of this invention can be prepared by .truncating the peptides of Fig. 9, for example, from the N-terminus to 1 delete the Y, tyrosine residue in SEQ 1D NOS. 21 93.

Such monomeric peptides can also be prepared by substitution of Y in the peptides of. lig. 9.

These molecules, demonstrate only binding activity in their 'monomeric' form, but exhibit agonist activity after dimerization with a linking compound such as PEG. Accordingly, the present method comprises identifying a monomeric peptide as .herein defined which does not demonstrate biological activity and dimerizing that antagonist in accordance with the pxrnt invention to obtain a cell-surface-receptor agonist in dimeric form. Contacting the appropriate cell-surface receptor with the thus formed dimer activates, i.e.

dimerizes such receptors and thus induces biological activity of the receptor. Such monomeric units as shown in Fig. 9 can be truncated from the N-terminus such as SCHFGPLTWVCK (SEQ ID NO: 18) to eliminate the tyrosine residue at position. A of the formula (X IX;,XX.XGPX,TWX,X. (SEQ ID NO: 19) or merely substituted with any of the remaining 19 naturally occurring amino acids or with other than a non-naturally occurring aromatic amino acid analog. In accordance with the present invention it has been determined that the.tyrosine residue at position A of the foregoing formula is critical to biological activity of the monomer peptide. Deletion or substitution of the tyrosine eliminates biological activity. When dimerized however the entity exhibits enhanced biological activity.- For example, tyrosine substituted in the formula YX 2 XaX.X,GPXXe (SEQ ID NO: 2) by p- -21iodohydroxyphenylalanine, p-fluorohydroxyphenylalanine, p-amino-hydroxyphenylalanine act as EPO-R monomer agonists but. substitution with threonine or alanine for tyrosine at position Y causes the monomer peptide to act as an EPO-R antagonist. However, when dimerized in accordance with the present invention, such dimers behave as EPO-R agonists. The monomeric peptide units identified at Fig. 9, for example, behave as EPO-R antagonists in the absence of tyrosine at position Y of the formula above. When such antagonists are dimerzied, the dimer behaves as an EPO-R agonist.

In a further embodiment of the present invention, pharmaceutical compositions comprising at least one of the dimers of this invention can be employed to therapeutically treat disorders resulting from deficiencies of biological factors such as EPO, GH, GM-CSF,' G-CSF, EGF, PDGF,-VEGF, insulin, FGF and the like. These pharmaceutical compositions may contain buffers, salts and other excipients to stabilize the composition or assist in the delivery of the dimerized molecules.

In a preferred embodiment, the present invention provides a method for treating disorders associated with a deficiency of EPO. The method is accomplished by administering at least one of the dimers identified herein for a time and under conditions sufficient to alleviate the symptoms of the disorder, i.e. sufficient to effect dimerization or biological activation of EPO receptors. In the case of EPO such 0 methodology is useful in the treatment of end-stage renal failure/dialysis; anemia, especially associated -22with.AIDS or chronic inflammatory diseases such as rheumatoid arthritis and chronic bowel inflammation; auto-immune disease; and for boosting the red blood cell count of patient when necessary, e.g. prior to surgery or as pretreatment to transfusion. The dimers of the.

present invention which behave as EPO agonists can be used to activate megakaryocytes.

Since EPO has been-shown to have a mitogenic and chemotactic effect on-vascular endothelial cells as well as an effect on central cholinergic neurons (see, Amagnostou et al. (1990) Proc. Natl. Acad. Sci.

USA 87:597805982 and Konishi et al. (1993) Brain Res.

609:29-35), the compounds of this invention can also be used to treat a variety of vascular disorders, such as promoting wound healing, growth of collateral coronary blood vessels (such as those that may occur after myocardial infarction), trauma, and post vascular graft treatment, and a variety of neurological disorders, generally characterized by low absolute levels of acetyl choline or low relative levels of acetyl choline as compared to other neuroactive substances e.g., neurotransmitters.

Accordingly, the present invention includes pharmaceutical compositions comprising, as an active ingredient, at least one of the peptide dimers of the present invention in association with a pharmaceutical carrier or diluent. The dimers of this invention can be administered by oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation) or transmucosal -23- (nasal, vaginal, rectal, or sublingual) routes of administration in dosage forms appropriate for each route of administration..

Solid dosage forms for oral administration include capsules, tablets, pill, powders, and granules.

In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch.

Such dosage forms can also comprise, as it normal 0 practice, additional substances other than inert diluents, lubricating, agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering, agents.

Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, with the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.

Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.

Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, -24- 1 emulsifying, and dispersing agents. They may be sterilized by, .fnsr example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other sterile injectable medium, immediately before use.

Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active.substance, excipients such as cocoa butter or a suppository wax. Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.

The dosage of active ingredient in the 5 compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient shall.be such that a suitable dosage form is obtained.

The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. Generally dosage.

levels of between 0.001 to 10 mg/kg of body weight daily are administered to mammals.

As can be appreciated from the disclosure above, the present invention has a wide variety of 5 applications. Accordingly, the following examples are offered by way of illustration, .not by way of limitation.

EXAMPLE 1 SDS-PAGE gels (10-20% gradient SDS-PAG plates, 84'x 70 x 1.0 mm, Integrated Separation Systems, Natick, MA) were stained with Coomasie Brilliant Blue R-250 (BioRad). A commercial preparation of activated difunctional polyethylene glycol (PEG-succinimidylpropionate, SPA2, MW ca. 3400) was purchased from Shearwater Polymers, Huntsville, AL as was the monofunctional reagent, methoxy-PEG-succinimidylproprionate, MW ca 5000. Peptide (SEQ ID NO: 8) and all other peptides were obtained from the Peptide Synthesis Facility RWJ-PRI, La Jolla, CA or-Quality Controlled Biochemical, Hopkinton MA. These peptides were cyclized via oxidation of their intramolecular cysteines, amidated at the C-terminus and mass confirmed by FAB-MS.

All were Ellman Reaction negative. Tris base was obtained from BioRad, Hercules, CA. (DPDPB) and trifluoracetic acid (HPLC grade) were obtained from Pierce Chemical Co., Rockford IL.

Mono-PEG conlucation of DeDtide GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 8) This example describes the preparation of mono-PEG conjugates of peptide (SEQ ID NO: using the monofunctional amine reactive polymer analog'm-SPA-PEG to be used as a control in experiments described herein.

The reaction was carried out with polymer in excess (ca.

3 fold) by resuspending 142.5 mg (0.0286 mmol, MW ca.

0 5000) of polymer in.4 ml PBS at pH 7.5 and adding :0 r-g peptide (SEQ ID NO: 8) (0.0095 mmol, MW 2092) dissolved -26in 1 ml of 0.1% trifluoroacetic ac-d. The mixture was incubated on ice for 20 hours. T'e reaction was subsequently adjusted to a final concentration 50 mM Tris by the addition of 1 M tris-HCl at pH 7.5. The reaction mixture was incubated on ice for one hour.

Analytical HPLC suggested that there.were two main reaction products of essentially equivalent magnitude which were not baseline resolved. Preparative

HPLC

(using the flatter gradient system described in Example 8) and conservative cuts resulted in czllection of two product peaks eluting at ca 44 and 47 minutes. After lyophilization, 24.8 mg, 16.5 mg of each species was recovered, respectively. Mass spectral analysis of these two species demonstrated centroid masses of -7092 S(peak 1) and 12036 (peak 2) indicating the coupling of one or two PEG molecules, respectively, to the peptide (Table I).

Tris inactivated dolymer. Tris inactivated polymer was formed by incubation of 5 mM SPA2 polymer dissolved in PBS (Gibco, Gaithersburg, MD) with tris-HCl, pH 7.5 added and used without further purification.

TABL E 1 Recovery Yield of Peptide Conjugation Reaction and Apparent Molecular Mass of Product I. No. Sequence Mass Conjugation Reagent Main Product Mass (centroid ni/z) YiCld of tlicorceical) 8 GTYSCIIFGPLTrIVCKPQGG 3 GGTYSCIF GPJTWCKPQ 0 Ac-GGTYSCIIFGPLTWVCKPQGO 4 GGLYACIfMPH'TWVCQPLRG R SCIIFGPLI'WVCK 2092 SPA2-PEG (HW ca. 34001 lu-SPA-PEOG hmW ca. 5000) 2133 2177 1375 GPA2-PEG SPA2-PEG S PA2 -P EG S PA 2-PEG 7834 7092 (pc-ak 1) 12036 (peak 2) 7862 7072 6326 54 37 -28- EXAMPLE 2 PEG dimerization of Deotide GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: 81 (lot #1f Examples 2 7 describe the dimerization.of various peptides described by the present invention.

The modification of peptide (SEQ ID NO: 8) was carried out by resuspending 25 mg (0.0071 mmol) of polymer in 4 ml PBS at pH 7.5, and adding a 3 fold molar excess of peptide (SEQ ID NO: 8) (0.0213 mmo.l, 44.5 mg, MW 2092) dissolved in 1 ml of 0.1% trifluoroacetic acid. The mixture was incubated on ice. After 3 hours of incubation, an additional 7.5 mg (0.0036 mmol) of lyophilized peptide was added, resulting in a final ratio of 3.5 moles of peptide per each mole of SPA2.

The mixture was incubated an additional 17 hours on ice.

The reaction mixture was adjusted to a final concentration of 50 mM Tris by the addition of 1 M tris- HC1 of pH 7.5 and incubated on ice for 1 hour. The sample was subjected to analytical and preparative HPLC as described in Example 8. After preparative HPLC and lyophilization, 38 mg of PEG dimer.was recovered. The theoretical yield for this experiment was 55 mg based on a calculated mass of 7600 mg/mmol for a yield of .69% (Table I).

EXAMPLE 3 PEG dimerization of Deotide GGTYSCHFGPLTWVCXPQGG (SEQ ID NO: 8) (lot #2) The modification of peptide (SEQ ID NO: 8) was carried out by resuspending 25 mg (0.0071 mmol) of polymer in 4 ml PBS at pH 7.5, and adding a ca. 3 fold molar excess of peptide (SEQ ID NO: 8) (0.0213 mmol, 45.8 mg, MW 2092) dissolved in 1 ml of 0.1% trifluoroacetic acid. The mixture was incubated on ice for 22 hours. At that time, the reaction was adjusted to a final concentration of 50 mM Tris by the addition of 1 M tris-HC1, pH 7.5. The reaction mixture was l-^ incubated on ice for 1 hour. The sample was subjected to analytical and preparative HPLC as described in Example 8. After preparative HPLC and lyophilization, 37 mg of PEG dimer was recovered. The theoretical yield for this experiment was 55 mg based on a calculated mass of 7600 mg/mmol for a yield of 68% (Table I).

1 EXAMPLE 4 PEG dimerization of Deotide GGTYSCHFGPLTWVCKPQ (SEQ ID NO: 13) The modification of peptide (SEQ ID NO: 13) was carried out by resuspending 11.2 mg (0.0033 mmol) of polymer in 2.5 nml PBS at pH 7.5, and adding a ca. 3 fold molar excess cf peptide (SEQ ID NO: -13) (0.010 mmol, mg, MW 1978) dissoclved in 0.25 ml of 0.1% trifluoroacetic acid. The mixture was incubated on ice for 20 hours. At that time, 0.25 ml of 1 M tris-HC1 at pH 7.5 was added. The reaction-mixture was incubated at 4°C for one hour. The sample was subjected to analytical and preparative HPLC as described in Example 8. The main preparative reaction product peak eluted at ca 43 minutes. After preparative HPLC and lyophilization, 13.3 mg of PEG dimer was recovered. The theoretical yield for this experiment was 24.42 mg based on a calculated mass of 7400 mg/mmol for a yield of 54% (Table I).

-31- 1 EXAMPLE PEG dimerization of DeDtide Ac-GGTYSCHFGPLTWVCKPQGG (SEQ ID NO: The modification of peptide (SEQ ID NO: was carried out by resuspending 10.5 mg (0.0031 mmol) of polymer in 2.5 ml PBS at pH 7.5, and adding a ca. 3 fold molar excess of peptide (SEQ ID NO: 20) (0.0094 mmol, mg, MW 2133) dissolved in 0.25 ml of 0.1% trifluoroacetic acid and the mixture incubated at 4°C for 28 hours. At that time, the-reaction as monitored by HPLC was estimated to be approximately 30% complete, the temperature was shifted to ambient and an additional 27 hour incubation provided no net increase in product.

Because of possible hydrolysis of the reactive polymer, an additional 5 mg of polymer was -added and the incubation was continued for an additional 16 hours. At that time, 0.25 ml of 1 M tris-HCI, pH 7.5 was added and 2 the reaction mix was incubated at 4 0 C for an additional 1 hour. The sample was subjected to analytical and preparative HPLC using a flatter gradient system as described in Example 8. The main preparative reaction product .peak eluting at ca 48 minutes. After preparative HPLC and lyophilization, 10.4 mg of PEG dimer was recovered. The theoretical yield for this experiment was 34.4 mg based on a calculated mass.of 7650 mg/mmol for a yield of 30% (Table I).

-32- EXAMPLE 6 PEG dimerization of Deotide (SEQ ID NO: 14) The modification of peptide (SEQ ID NO: 14) was carried out by resuspending 2.6 mg (0.00076 mmol) of polymer in 3.0 ml PBS at pH 7.5 and adding a ca. 3 fold molar excess of peptide (SEQ ID NO: 14) (0.00229 mmol, .mg, MW 2177) dissolved in 0.1 ml of 0.1% trifluoroacetic acid. The mixture was incubated on.ice for 26 hours.

At that time, 0.25 ml of 1 M-tris-HCl at pH 7.5 was added. The reaction mixture was incubated at 4 0 C for 1 hour. The sample was subjected to analytical and preparative HPLC using the flatter gradient system described in Example 8. The main preparative reaction product peak eluted at ca 46 minutes. After preparative HPLC and lyophilization, 2.2 mg of PEG dimer was recovered. The theoretical yield for this experiment was 24.42 mg based on a calculated mass of 7400 mg/mmol for a yield of 37% (Table I).

-33- 1 EXAMPLE 7 PEG dimerization of DeDtide (SEQ ID NO: 18) The modification of peptide (SEQ ID NO: 18) was .carried out by resuspending -1.2 mg (0.00036 mmol) of polymer in 0.5 ml PBS at pH 7.5, and adding a ca. 3 fold molar excess of the peptide (0.0011 mmol, 1.5 mg, MW 2177) dissolved in 0.05 ml of 0.1%-trifluoroacetic acid.

The mixture was incubated on ice for 20 hours. At that time, 0.1 ml of 1 M tris-HCl at pH 7.5 was added. The reaction mixture was incubated at 4 0 C for 1 hour. The sample was subjected to purification using an analytical HPLC system as described in Example 8. The main reaction product peak eluted at ca 38 minutes. After preparative HPLC and lyophilization, 1 mg of PEG dimer was recovered. The theoretical yield for this experiment was 2.2 mg based on a calculated mass of 6150 mg/mmol for a yield of 45% (Table I).

I

-34- EXAMPLE 8 Analytical and Preparative HPLC analysis The accumulation of the.dimers described above in Examples 1-7 was monitored by analytical reverse phase HPLC. The analysis was carried out using a Vydac C-18 Protein-Peptide column (0.46 x 25 cm, part no.

0 218TP54) and a Rainin Gradient HPLC system fitted with a Dynamax dual wavelength detector. At injection, the column was equilibrated in 0.1% TFA in dH 2 O and was developed with a 45 minute linear gradient (0-100%) of acetonitrile (ACN) containing 0.1% TFA beginning at minutes after injection. The flow rate was held Sconstant at 1 ml/min. Under these analytical conditions, the SPA2 polymer and tris inactivated polymer did not appear to bind the column while a major reaction product with a retention time (37 minutes) was identified (Figure Peptide (SEQ ID NO: 8) demonstrated a retention time of 35 minutes and the excess peptide utilized in the reaction was clearly distinguished from the nascent reaction products.

The main product reaction product peak was purified by preparative reverse phase HPLC on the same chromatographic system using a Vydac C-18 Protein- Peptide column (2.2 x 25 cm, part no. 218TP15022).

Injection of the reaction mix (6 ml) occurred with the column equilibrated at 80:20, HO:ACN (both containing 0.1% TFA) at a constant flow rate of 8 ml/min. After a 20 minute wash, the column was developed tv application of a linear gradient of 100% ACN/0/1% TFA over minutes. The major product peak eluting at 48 minutes was collected and lyophilized (Figure These elution conditions were subsequently modified to improve the resolution of some conjugation products peptide (SEQ .ID NO: 20), mPEG-peptide (SEQ -ID NO: peptide (SEQ ID NO: 14) from reaction by products. This was accomplished by application of a flatter linear gradient of 20-80% B over 60 minutes. The variation in retention time due to-different peptides and elution condition is described as part of each synthesis example. The materials recovered from the main product peak from each reaction were -subsequently analyied by analytical reverse phase HPLC, MALDI-TOF mass spectrometry,

EPO

competitive binding potential and for in vitro bioactivity.

The activated PEG used in these experiments has an approximate molecular weight of 3400 and has amine reactive succinimidyl groups on either end of the difunctional linear polymer. This reactivity was 2 employed to couple two equivalents of peptide (SEQ ID NO: 8) (MW= 2092) to the polymer with the concomitant liberation of two succinimidyl moieties resulting in a dimeric product as shown in Scheme I. Peptide (SEQ ID NO: 8) contains two potentially reactive amines, one at the N-terminus of the peptide and one in the side chain of the single lysine within th peptide sequence, so that a number of different connectivities between the two molecules was possible.

MALDI-TOF mass spectral analysis was supportive of the presence of the expected dimeric product (Figure 3) as indicated by a predominant.species.

-36w4ith a centroid mass of 7661. This data shows that the dime---ric product -described in the present invention was produced using the methods described herein.

-37- EXAMPLE 9 EBP (EPO Bindino Protein) Dimerization This example demonstrates the interaction of peptide (SEQ ID NO: peptide (SEQ ID NO: 16), peptide (SEQ ID NO: 18) and peptide (SEQ ID NO: 13) with EPO binding protein (EBP) using a bifunctional sulphydryl reactive crosslinker, (2'-pyridyldithio)propionamidoj]btane

DPDPB.

To explore the interaction of peptide (SEQ ID.

NO: 8) with EBP, a bifunctional sulphydryl reactive crosslinker (DPDPB) was used in an attempt to stabilize a mimetic dependent dimeric structure. Control experiments demonstrated that the crosslinker does not inactivate the EPO binding potential of EBP or the proliferative properties of peptide (SEQ ID NO: As shown in Figure 4, a dimeric EBP product was formed by co-incubation of the peptide, peptide (SEQ ID NO: 8), DPDPB and EBP. This data shows the ability of the peptide (SEQ ID NO: 8) to mediate formation of a soluble receptor dimer. To further explore this question, peptides (SEQ ID NO: 13), (SEQ ID NO: 16) and (SEQ ID NO: 18) were examined for their ability to mediate dimerization. As shown in Figure 4, lanes 7A and 8A, when peptide (SEQ ID NO: 13) was truncated at the carboxyl terminus, it retained good in vitro bioactivity and improved in vivo bioactivity, resulting in a 0 crosslinking signal similar to peptide (SEQ ID NO: 8).

However, peptide (SEQ ID NO: 18) did not appear to stabilize the dimerization signal (Figure 4, lanes 9A -38and 10A) whereas peptide (SEQ ID NO: 16) (Figure 4, lanes 5A and 6A) gave a scrong dimerization band. These two peptides differ by a single N-terminal tyrosine residue and display a similar-profile in the in vitro proliferation assay with peptide (SEQ ID NO: 18) being inactive. Peptide (SEQ ID NO: 16) has an EDso of 3 WM on murine.receptor cells. Both-peptides have similar ICso values indicating that they both retain binding 0 activity. These results demonstrate that EBP dimerization is a property of the EPO peptide series and that the presence of the tyrosine is critical for this activity and that this corresponds to in vitro bioactivity.

-39- EXAMPLE -IMMOBILIZED EBP BASED r[=I-1EPO COMPETITION

BINDING

ASSAY

This study examined the binding capacity of the EPO PEG dimers to bind EPO receptors.

The extracellular domain of the human erythropoietin receptor (EPO binding protein, EBP) was expressed and overproduced in E. coli. As with many other recombinant eukaryotic proteins produced in E.

coli, the protein appeared as! an insoluble product in laboratory scale fermentations and was refolded and purified to obtain active protein. EPO binding protein produced by this method contains one free sulfhydryl group which can be modified without effecting the solution phase binding of ligand. In order to immobilize the EPO binding protein for equilibrium binding analysis and for competition binding assay, the EPO binding protein was covalently attached to agarose beads.

The iodoacetyl activation chemistry of Sufolink beads (Pierce Chemical Co, Rockford, IL) is specific for free thiols and assures that the linkage is not easily reversible. EBP-Sulfolink beads were made as follows: SulfoLink gel suspension (10 ml) was mixed with of coupling buffer (40 ml: 50 mM Tris, pH 8.3, 5 mM EDTA) and the gel was allowed to settle. The supernatant was removed and the EPO binding protein (0.3-1 mg/ml in coupling buffer) to be bound was added directly to the washed beads. The mixture was rocked gently for 30 minutes at room temperature and the beads were allowed to settle for 1 hour at room temperature.

The supernatant was removed and retained. The beads S were washed twice with 20 ml of coupling buffer. The washes were recovered as well. The beads were then treated with 20 ml of 0.05 M cysteine for 30 minutes at room temperature to block unbound sites. Finally, the beads were washed with 50 ml of 1-M NaC1, then with 0 ml of PBS, and resuspended in 20 ml of PBS and stored at 4 0 C. The amount of EBP which was covalently bound to the beads was determined by comparing the OD=.o of the original EBP.solution to the total OD.

o recovered in the reaction supernatant and the two 20 ml washes.

Typically, 40-60% of the applied EBP remains associated with the beads.

Binding assays were initiated by the addition of .EPO binding protein beads (50 pl) to individual reaction tubes. Total binding was measured in.tubes containing 0.3-30 nM ["I])EPO (NEN Research Products, Boston MA, 100 MCi/pg). For.determination of non-specific binding, unlabelled EPO was added at a level of 1000 fold in excess of the corresponding [I"I)EPO concentration. Each reaction volume was brought to 500 pl with binding buffer (PBS/0.2% BSA).

The tubes were incubated for five hours (a time period experimentally determined as adequate for the establishment of equilibrium) at room temperature with gentle rocking. After five hours, each reaction mixture 0 was passed through a 1 ml pipet tip plugged with glass wool. The tubes were washed with 1 ml wash buffer

(PBS/

BSA) and this volume as well as 2 additional 1 ml -41washes were passed through the pipet tip and collected for determination of the free EPO concentration.

Equilibrium binding analysis of the specific associationof with EPO mimetic binding proteins immobilized on these agarose beads indicates a Kd of nM 2 based on a linear transformation (Scatchard) of the binding isotherm (Figure Competitive binding analysis assays of 0 candidate peptides and dimer peptides were perfcrmed as outlined below. Individual peptides were dissolved in DMSO to prepare a stock solution -1 mM. Dimer peptides were contained within PBS at a concentration of 5 mM.

All reaction tubes (in duplicate) contained 50 pL of EBP beads, 0.5 nM ["I]EPO and 0-500 IM peptide in a total of 500 pL binding buffer.

The final concentration of DMSO was adjusted to 2.5% in all peptide assay tubes. At this concentration DMSO has no detectable effect since an examination of the sensitivity of the assay to DMSD demonstrated that concentrations of up to 25% DMSO (V/V) had no deleterious effect on binding. Non-specific binding was measured in each individual assay by inclusion of tubes containing a large excess of unlabelledEPO (1000 nM). Initial assay points with no added peptide were included in each assay to determine total binding. Binding mixtures were incubated overnight at room temperature with gentle rocking. The beads were then collected using Micro-columns (Isolab, Inc.) and washed with 3 mL of wash buffer. The columns containing the washed beads were placed in 12 x 75 mm glass tubes and bound radioactivity levels determined in -42a garma counter. The amount of bound ['"I]EPO was expressed as a percentage of the control (total=100%) binuing and plotted-versus the peptide concentration after correction for non-specific binding. The ICo was defined as the concentration of the analyte which reduced the binding of 1 I]EPO to the EBP beads by All data are reported as relative to peptide (SEQ ID NO: which demonstrated an ICso of 5 iM.

Competitive binding analysis revealed an ICso !0 of 20 pM for the purified dimer, a value four fold greater than peptide (SEQ ID NO: 8) in the same assay (Figure 5 and Table II). Polymer alone, which was inactivated by treatment with Tris-HCl, demonstrated a detectable competition binding signal but this signal was modest at the IC.o of the PEG-peptide (SEQ ID NO: 8) dimer.

43 Table II Binding and Cell. Proliferation Studies Compound Relative Binding' EPO-ED-v (WM* ttuncated.

muine- receptor human receptor seaildno8 1. 0.1. 0.09 SA.P2 tris inact. Polymer 60 I

I

SeqidnoS, covalent dixner#l 4 0.02. (lox) 0.0015 (6Ox) SeqoudoS, covalent-dimer-#2 3 0.01 (lox) 0.002 (4sx) secidnl3 1. .80.02 Seqidno:l3, covalent dimer 3 0.01 (8x) 0.002 (lOX) Seqidnc2o (N-acetyl) 4 0.03 0.06 Seaidno2O, covalent clime= 12 0.2 0.05 Seqidno3.4 (terminal NF,) 0.6 0.1 0.08 Seqid~nol4, covalent dimzer ND 0.006 (16X) 0.001 (BOX) SAmount required to achieve the half maximal level of IEPO dependent proliferation (11pM) 'Binding relanve to RWJ-6123 *Note that all peptdes* are cyclic and were analyzed as COOH terminal amide (-ONHS -44- EXAMPLE 11 EPO DEPENDENT CELL PROLIFERATION

ASSAYS

This example shows the improved potency of PEG-EPO peptide dimers to EPO receptors in human and murine cell lines.

Cell line FDC-PI/ER, an EPO-dependent line expressing the murine EPO receptor, was grown and maintained as described previously (Carroll et al.

1991). Also employed was cell- line FDC-Pl/trER expressing a functional truncated human EPO receptor (missing the-C-terminal 40 amino acids). Both'cell lines exhibit EPO dependent cellular proliferation.

Briefly, cells were maintained in RPMI 1640 media (Gibco/BRL) containing 10% heat-inactivated fetal calf serum and 10 units/ml of recombinant human EPO. For the cellular proliferation assay, FDC-P1/ER or FDC-Pl/trER 0 cells were grown to stationary phase, centrifuged, washed with RPMI 1640 media (no EPO), and plated in EPO minus media for 24 hr.

After 24 hours, the cells were counted, resuspended at 800,000 cells/ml and dispensed at 40,000 cells/well. Stock solutions of the peptide dimer (5 mM in PBS) and peptide (10 mM in DMSO) were prepared and dispensed in triplicate to final- concentrations of 1 x 10-0 M through 1 x 10-' M and adjusted to a final volume of 0.2 ml. Final DMSO concentrations of 0.1% maximal) orless were found to have no cellular toxicity or stimulatory effects. A standard dcse response curve was generated with each assay series.

After a 42 hr incubation at 37 0 C (ca. 2 cell doublings) L~Ci/well of 3 H] thymidine was added and the incubation continued for 6 hr at which time the cells were harvested and counted to assess ['H]thymidine incorporation as a measure of cell proliferation.

Results are expressed as the amount of peptide or dimer peptide necessary to yield one half of the maximal activity obtained with recombinant EPO.

As shown in Figure 5 and Table II, the initial i0 lot of PEG-peptide (SEQ ID NO: 8) dimer demonstrated EDo, values of 0.01 pm and 0.0015 pm in EPO responsive cell lines containing the murine or human EPO receptor, respectively. In both cell lines, the parent peptide, peptide (SEQ ID NO: .demonstrated an ED=, of 0.1 pM, indicating an increase in potency of 10 fold in the murine receptor line and almost 60 fold in the human receptor containing cells. Thus, the dimer was clearly more potent in murine and human lines than the peptides themselves. This was confirmed by generation of a second synthesis lot of PEG-peptide (SEQ ID NO: 8) dimer which resulted in a 10 and 45 fold increase in potency in the murine and human lines, respectively. Polymer alone, which was inactivated by treatment with Tris-HC1, demonstrated no activity in the cell proliferation assay.

A second EPO mimetic peptide, peptide (SEQ ID NO: 13), with the sequence GGTYSCHFGPLTWVCKPQ, was also subjected to a similar PEG dimerization protocol as that described above for peptide (SEQ ID NO: The dimer 3O Sproduct of PEG-peptide (SEQ ID NO: 13) is also r.ore active than the unconjugated parent compound (Table II).

-4 6 1 Both of these dimner peptides have ultimate

ED

5 0 values near O.OO2g.M. In sipite of this -more mode.- iceae the experimental evidence .clearly indicates that the dimerization. of these peptides with PEG results in

I

-47- EXAMPLE 12 To further examine -the connectivity of the peptides of the present invention to PEG, peptide molecules, which contained only an internal lysine group were used peptide (SEQ ID NO: 8) analog acetylated at the N-terminus peptide (SEQ ID NO: 20) and a sequence analog peptide (SEQ ID NO: 14) which only had a reactive N-terminal amine were PEG dimerized. In vitro proliferation data of these compounds suggest that potential dimerization through.the free amino terminus has the most profound effect on bioactivity giving rise to a species about 80 fold more active than the monomeric parent peptide (SEQ ID NO: 14) dimer.

Conjugation through the lysine side chain had no real effect on activity peptide (SEQ ID NO: 20) as did mono- PEG or di-PEG conjugation (Table III). This data indicates that the creation of a head to head dimer (both peptides attached through the N-terminus) using a 2 PEG linker greatly enhances the potency of EPO peptides and approaches a level almost two logs greater than the free parent peptide. Further, this effect was not observed upon simple covalent attachment of linear PEG to peptide (SEQ ID NO: 8) indicating that dimerization is a critical determinant for this increased activity.

Page 48 Table H!l Table MI. Binding and Cell Proliferation Studies mPEG/SEQ. ED. NO. 8 Compound Relative Bindine EPO ED Murine Truncated Receptor Human Receptor: SEQ.- M. NO. 8t .100 mPEG/SEQ. ED. No. 8, peak #11 60 2 0.1 mPEG/SEQ. IJD. NO. 8, peak #2 >40 10.4 Amount required to achieve the half mnaximxal-level of Epo dependent Proliferation (1 1pM) *ND =Not deterimiined 2 IA Active Binding relative to SEQ. ID). No.8s Note that all peptides are Cyclic and were analyzed as COOH terminal amides

-CONH

2 -49- EXAMPLE 13 Polvcvthemic Exhvoosic Mouse Bioassav.

S This study demonstrates the ability of peptide (SEQ ID NO: 8)/PEG-dimers to retain in vivo bioactivity.

Peptides were assayed for in vivo activity in the polycythemic mouse bioassay adapted from the method described by Cotes and Bangham (1961), Nature 191: 1065- 1067. BDF1 mice were allowed to acclimate to ambient conditions for 7-10 days. Body weights were determined for all animals. Low weight animals (<15 grams) were not used. Mice were introduced to hypobaric chambers with a 24 hour conditioning cycle consisting of 0.40% 5 0.02 atm. for 18 hours followed by 6 hours at ambient pressure for a total of 14 days. Following the 14 day period, mice were placed in ambient pressure for 72 hours prior to dosing. Test samples or recombinant Human Erythropoietin (rHuEPO) standards were diluted in an assay vehicle consisting of Phosphate Buffered Saline (PBS)-0.1% Bovine Serum Albumin (BSA). Peptide sample stock solutions (excluding peptide dimers) were first solubilized in dimethyl sulfoxide (DMSO). Control groups included one group of vehicle alone, and one group of (DMSO) at final concentration of 1%.

Each dose group contained 10 mice. Mice were injected subcutaneously (scruff of neck) with 0.5 ml of the appropriate sample. Forty eight hours following the sample injection, the mice were administered an intraperitoneal injection of 0.2 ml of ["Fe] S (approximately 18.0 i.i lliCuries/milligram, Dupont, NEN) and 0.75 microCuries/M1use.

Mouse body weights were determined twenty four hours following administration and the mice were S sacrificed forty eight hours following the injection. Blood was collected from each animal by cardiac puncture and hematocrits were determined (heparin was used as the anticoagulant). Each blood sample (0.2 ml) was analyzed for 9 Fe] incorporation using a Packard gamma counter. Non-responder.mice (i.e.,those mice with radioactive_.incporporation less than the negative control group) were eliminated from the appropriate data set. Mice that had hematocrit values less than 53% were also eliminated.

This assay examined the ability of an exogenously administered compound to induce new red blood cell synthesis, or in other words to function as EPO or an EPO mimetic. The results are derived from sets-of 10 animals for each experimental dose. As shown in Figure 7 and Tabla IV, the data suggests that on a mole equivalent basis, peptide (SEQ ID NO: 8)/PEG-dimer is about 10 fold more active than peptide (SEQ ID NO: 8) monomer. These results are consistent with in vitro results in which increased potency values of 10 fold was observed on murine EPO-R bearing cells.

TABLE IV Tb 4. Embpoxi Mouse Biuy Smdy of PEG Dimer Acmviry CAmpoAnd Amount rmuid for equivaecry to .025U EPO (nnot) n=1rO Seq. I.D. No. 8 -51- EXAMPLE 14 This example shows that an inactive truncation analog of peptide (SEQ ID NO: which lacks the critical tyrosine peptide (SEQ ID NO: 18), (SCHFGPLTWVCK), can be converted to an agonist on the human EPO receptor cell line by PEG dimerization.* In this experiment, a 10 5 M concentration of the parent peptide had no activity above background while the dimeric peptide exhibited a level of proliferation twice as many cpm as background. As shown in Figure 8, the peptide alone (open squares) did not induce proliferation of the EPO responsive cells but upon PEG dimerization (open diamonds)'a significant agonist effect was observed. Approximately twice as many cpm incorporated over non-stimulated cells at 10 M added peptide dimer. The replicate error bars represent the standard deviation of three assay points per concentration of peptide or peptide dimer.

1 Exhvpoxic oDlvcvthemic mouse bioassay: PEG dimer and the monomer parent peptide RWJ 61118 were compared in the exhypoxic mouse bioassay (Table This peptide exhibited an fold increase in in vitro activity upon dimerization. Murine studies (in vivo) of the activity of the dimer compared to the monomer peptide revealed a 250 fold increase in activity of RWJ 61718 upon dimerization. Cellular proliferation studies on this dimer peptide in murine receptor containing cells demonstrated a 16 fold increase over the monomer indicating that the 250 fold increase in vivo might be attributable to other factors such as altered metabolism or prolonged circulatory half-life which occur upon PEG dimerization. of the peptide sequence. -Thus, in addition to the effect of dimerization alone, the PEG modification has an effect which impacts in vivo activity and may be specific to individual peptide sequences.

Cell associated EPO receptor competition bindino assay.

A competitive binding analysis of the ability of selected monomer peptides and their cognate dimer products to compete with radiolabelled EPO binding for cell associated h-man EPO receptors was performed Erythropoietin Receptor Competition Binding Analysis was preformed as follows. TF-1 cells were maintained in RP-I 1640, fetal calf serum, 1% L-glutamine, 1% penicillin, 0.1% streptomycin and 1 ng/ml of GM-CSF.[125]-EPO was obtained from NEN Research Products. Cells were centrifuged and washed 1 x with binding buffer (RPMI 1640, 5% BSA, 25 mM Hepes, pH 7.5, 0.02% sodium azide) resuspended in binding buffer, and counted using trypan lue as an indicator of viability. Each reaction contained approximately x 10 s cells, [125]-EPO (0.5 nM), no competitor or peptide or dimer preparation in a final volume of 200 l. The binding reactions (in duplicate) were incubated overnight at 4'C. Following binding, the tubes were centrifuged at 12,000 rpm for 1 min at 4'C in a 53 12 refrigerated centrifuge. The supernatant was removed, the cell pellet resuspended in 100 pi of binding buffer, and the cell suspension layered onto 0.7 ml of bovine calf serum. The tubes were centrifuged at 12,000 rpm for 5 min at 4'C, the supernatant was removed, the bottom of the tubes snipped off, and the cell pellets counted in a Micromedic ME plus gamma counter. Non-specific binding was determined by incubating cells with [125]-EPO and a 100-fold excess of non-radioactive EPO. These data demonstrate increases in apparent binding competitive affinity of 3.0 fold, 3.2 fold and fold for peptides RWJ 61233, RWJ 61596_and RWJ 61718, respectively (Table In vivo proliferation studies with these peptides and their dimer derivatives reveal increases in potency of ea. 50 fold, fold and 80 fold, respectively, indicating that the magnitude of increased binding affinity is exceeded by the functional potency of the peptide for two of the three species. Thus, the effect of dimerization and subsequent increase in activity may be one in which the efficiency of receptor stimulation is improved by limiting the lateral diffusion of the receptors away from a binding event.

Peptide dimerization therefore likely results in entropic rather than enthalpic gains upon mimetic ligand-receptor association for some peptide dimer sequences.

Unlike the EBP-bead EPO competitive binding assay where peptide dimerization negatively impacted the ability of PEG dimer peptides to compete for receptor binding, the ability to compete for cell associated -receptors is increased by dimerization. This may be due to the ability of the cell associated-receptor to dimerize while the immobilized EBP monomer likely cannot.

Conversion of inactive to active peptide RWJ 61177 was further studied. An improved and expanded study was performed which confirmed our earlier observation of conversion to an active pectide (Figure 6, Panel D).

54 TABLE IV. EXHYPOXIC MOUSE BIOASSAY STUDY OF PEG DINMER

ACTIVIT

Compound Amount required for equivalency to 0.025 U EPO (nz-nol) n= RWJ 61233 (seq. ID#8) SAP2I6 1233, covalent dixner RWJ 61718 (seq. ID 914) SAP2161718, covalent dixner 0.28 0.07 55 TABLE V, EPO COMPETITIVE BINDING ANALYSIS OF CELL ASSOCIATED

RECEPTORS

Compound

IC

50

(PM)

RWJ 61233 (seq ID #8) SAF2/6 1233, covalent dimer RWI 61718 (seq IDA 14) SA.P2/61718, covalent dirner 3.8 0.28 0.07

I

56 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Johnson, Dana L Zivin, Robert A (ii) TITLE OF INVENTION: AGONIST PEPTIDE DIMERS (iii) NUMBER OF SEQUENCES: 93 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Frank S. DiGiglio STREET: 400 Garden City Plaza CITY: Garden City STATE: New York (E).COUNTRY: U.S.A..

ZIP: 11530 COMPUTER READABLE FORM MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 CURRENT APPLICATION DATA: APPLICATION NUMBER: US 08/484,135 FILING DATE: 07-JUN-1995

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: DiGiglio, Frank S REGISTRATION NUMBER: 31,346 REFERENCE/DOCKET NUMBER: 9594 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (516) 742-4343 TELEFAX (516) 742-4366 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Peptide 57 LOCATION: OTHER INFORMATION: /note= "Xaa(Posl) can be C,AA-nmino-ybromobutyhc acid or Hoc; Xaa(Pos2) can be RHL or Xaa(Pos3) can be MF or I; Xaa(Pos6) can be any one of the 20 L-amino acids or dhe stereoisomeric D-amino acids; Xaa(Pos9) can be D,IL or V; and Xaa(PoslO) can be CA,-amino-y-brornobutrfic acid or Hoc, provided that either Xaa(Pos I) or Xaa(Pos 10) is C or Hoc" (xi) SEQUENCE DESCRIP'TION: SEQ ID NO: 1: Xaa Xaa Xaa Gly Pro Xaa Thr Trp Xaa Xa 1 5 ()INFORMATION FOR SEQ ID NO:2:- SEQUENCE CHARACTEPISTICS: LENGTIH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Peptide LOCATION: 12 OTHER DNFORMATION: /note= "Xaa(Pos2) and Xaa(JPos8) can be any one of the 20 L-aniind acids; Xaa(Pos3) can be C,A.i-amino-y-, bromobutyric acid or Hoc; Xaa(Pos4) can be RB3,L or W; can beKF or I; Xaos1) canbe DEILorV; and Xaa(Pos 12) can be C,AA-arnino-y-bromobutyric acid or Hoc, provided that either Xaa(Pos3) or Xaa(Posl2) is C or Hoc" (xi) SEQUENCE DESCRIPTION: SEQ ID NO0:2: Tyr Xaa Xaa Xaa Xaa Gly Pro Xaa Thr Trp.Xaa Xaa 1 5 INFORMATION FOR SEQ ID NO:3:.

SEQUENCE CHARACTERISTICS: LENGT1 16 amino acids TYPE: amino acid STRANDEDNESS. singli TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAMIE/KESY: Peptide LOCATION: 1..16 OTHER INFORMATION: Inote= "Xaa(PosI1), Xaa(Pos Xaa(Pos9), 58 Xaa(Posl4), Xaa(Posl5) and Xaa(Posl6) can be any one of L-amino acids; Xaa(Pos4) can be C,Ai-aniino-y-bromobutyric acid or Hoc; Xaa(Pos5) can be R-L or W;Xaa(Pos6) can beMF orI; Xaa(Pos 12) can be D,E,IL or V; and Xaa(Pos 13) can be C,Aiamino-y-bromobutyric acid or Hoc, provided that either Xaa(Pos4) or XAa(Pos13) is CorHoc" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Xaa Tyr Xaa Xaa Xaa Xaa Gly Pro Xaa Thr Trp, Xaa Xaa Xaa Xaa Xaa 1 5 10 1s INFORMATION FOR SEQ ID NO*4 Wi SEQUENCE CHARACTERISTICS: LENGTH.- 16 ammno acids.

(B3) TYPE: amino acid STRANDEDNESS: single.

TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO*4 Xaa Tyr Xaa Cys Xaa Xaa Gly Pro Xaa Thr Trp Xaa Cys Xaa Xaa Xaa 1 5 10 INFORMATION FOR SEQ ID SEQUENCE CHARACTEPJSTICS: LENGTH: 16 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Peptide LOCATION: 16 OTHER INFORMATION: /note= "Xaa(Posl), Xaa(Pos3) and Xaa(Posl6) ca eany one of the 20 L-amino acids-, Xaa(Pos5) can be R or H Xaa(Pos6) can be F or K- Xaa(Pos9) can be ITM or V;, Xaa(Pos12) can be D or Xaa(Posl4) can be GK.,QRS or T; can be AGPR or Y" SEQUENCE DESCRIPTION: SEQ ID Xaa Tyr Xaa Cys Xaa Xaa Gly Pro Xaa Tbr Trp Xaa Cys Xaa Xaa Xaa 1 5 10 59 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERSTICS: LENGTH: 16 amino acids TYPE: -amino acid STRANDEDNESS: single TOPOLOGY: linear (ui) MOLECULE TYPE: peptide (Ix) FEATURE.

NAME/KEY: Peptide LOCATION: 16 OTHER INFORMATION: /note= 'Xaa(Pos I can be DE,LNS,T or V; Xaa(Pos3) can be AHXLMS or T; Xaa(Pos5) can be R or II Xaa(Pos;6) can be F or M; Xaa(Pos9) can be ILTM or V; Xaa(Posl2) can be D or V; Xaa(PosI4) can be K,,S orT;is P and Xaa(Posl6) can be any one of the 20 L-amino acids" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Xaa Tyr Xaa Cys Xaa Xaa Gly Pro Xaa Thr Trp Xaa Cys Xaa Xaa Xaa 1 5 10 INFORMATION FOR SEQ ID NO:7: Wi SEQUENCE CHARACTERISTICS: LENGTH 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SEQUENCE DESCRIPTON: SEQ ID NO:7: Gly Gly Leu Tyr Leu Cys Arg Phe Gly Pro Val Thr Trp Asp Cys Gly 1 5 10 Tyr Lys Gly Gly R4FORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: ammno acd STRANDEDNESS: single TOPOLOGY: linear 60 (ii) MOLECULE T YPE: peptide SEQUENCE DESCRIPTION: SEQ ID NO:8: Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 Pro Gin Gly Gly INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE-TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Gly Gly Asp Tyr His Cys Arg Met Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 Pro Len Gly Gly INFORMATION FOR SEQ ID) SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide (xm) S EQUENCE DESCRIPTION: SEQ ID NO: Val Gly Asn Tyr Met- Cys His Phe Gly Pro Ile Thr Trp Val Cys Arg 1 5 .10 Pro Gly Gly Gly INFORMATION FOR SEQ ID NO:11: 61 SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ M. NO: 11: Gly Gly Val Tyr Ala Cys Arg Met Gly Pro fle Tbr Trp Val Cys Ser 1 5 10 Pro Leu Gly Gly INFO0RMATION FOR SEQ ID) NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide Wx) SEQUENCE DESCRIPTON: SEQ ID NO: 12: Val Gly Asn Tyr Met Ala HsMet Gly ProlIle Thr Trp Val Cys Arg 1 5 10 1 Pro Gly Gly INFORMATION FOR SEQ NQ: 13: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid .STRANDEDNESS. siurfle TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xd) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 Pro Gin 62 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH* 20 amino acids.

(B3) TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION:- SEQ ID NO:14: Gly Gly Leu Tyr Ala Cys His Met Gly Pro Met Thr Trp Val Cys Gin 1 5 10 Pro Leu.Arg Gly INFORMAT1ON FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH- 22 amino acids TYPE: amino acid STh.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTON: SEQ ID NO: Thr Ile Ala Gin Tyr Ie Cys Tyr Met Gly Pro Glu Thr Trp Glu Cys 1 5 10 Arg Pro Ser Pro Lys Ala INFORMATION FOR SEQ ID NO: 16 SEQUENCE CHARACTERISTICS: LENGTI-L 13 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Thr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys 1 5 63 INFORMATION FOR SEQ IIDNO: 17:- @.)SEQUENCE CHARACTERIISTICS: LENGTH: 1I amino acids TYPE' amino acid: STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptidd SEQUTENCE DESCRIPTON: SEQ ID NO: 17: Tyr Cys His Phe Gly Pro Leu Thr Trp Val Cys INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids.

TYPE: amino acid STR ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xn) SEQUENCE DESCRIPTION: SEQ ID NO: 18:, Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys 1 5 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTRISTCS: LENGTHI 12 amino- acids TYPE: amino acid STRANDEDNESS:. single TOPOLOGY: linear (ii) MOLECULE TYPE: pept ide (ix) FEATURE: NAME/KEY: Peptide LOCATION: L.12 OTHER INFORMATION: /note= "Xaa(Pos 1) can be any one of the 20 Lamino acids; except that Xaa(Posl) may or may not be Y and Xaa(Posl) may be any non-naturaily occurring aromatic acid analog when Xaa(Posl) is Y. Xaa(Pos2) and Xaa(PosS) can be any one of the 20 L-amino acids; Xaa(Pos3) -can be C,Aia-amino-y-bromobutyric acid or Hoc; Xaa(Pos4) can beRHLL or W; Xaa(PosS) can beMNUor 1; Xaa(Posll) can be DEIL or V and Xaa(Posl2) can be C,Ai- 64 arnino-y-bromoburyxic acid or Hoc provided that either Xaa(Pos3) or Xaa(Posl2) is C or Hoc"i (xi) SEQUENCE DESCRIPTON: SEQ IM NO: 19: *Xaa Xaa Xaa Xaa Xaa Gly Pro Xaa Thr Trp Xaa Xaa .1 5 ()INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRUPTON: SEQ ED Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr.Trp Val Cys Lys 1 5 10 Pro Gin Gly Gly INFORMATION FOR SEQ ID NO:2 1: SEQUENCE CHARA CTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Gly Gly Thr Tyr Arg Cys Ser Met Gly Pro Met Thr Trp Val Cys Leu 1 5 10 Pro Met Gly Gly INFORMATION FOR SEQ MD NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 6-5 Cii) MOLECULE TYPE: peptide (xi) -SEQUENCE DESCRIPTION: SEQ ID N0:22: Gly Gly Met Tyr Ser Cys. M~g Met Gly Pro Met Thr Trp Val Cys Gly 1 5 -10 Pro Ser Gly Gly INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single.

TOPOLOGY: linear (ii) MOLECULE TYPE -peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Gly Gly Tr Aa Trp Cys Arg Met Ply Pm Ile Thr Trp Val Cys Ser 1 5 10 Ala His Gly Gly INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS:. single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: SEQ ID NO:24: Gly Gly Met Tyr Ser Cys Mrg Met Gly Pmo Met Thr Trp Val-Cys ile *1 .5 10 Pro Tyr Gly Gly INFORMATION FOR SEQ ID N0:25: 66.'- SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Gly Glu Tyr Lys Cys Tyr Met Gly Pro Ile Thr Tip Val Cys Lys 1 5 10 Pro Glu Gly Gly INFORMATION FOR SEQ ID N0:26: SEQUENCE CHARACTERISTICS: LENEGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ui) MOLECULE TYPE: pepude (xi) SEQUENCE DESCRIPTON: SEQ I.D NO:26: Gly Gly As p Tyr Thr Cys Arg Met Gly Pro Met Thr Trp Ile Cys Thr 1 5 10 Ala Thr Gly Gly INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTIh: 20 amino acids TYPE: amino acid STRANqDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide.

SEQUENCE DESCRIPTION: SEQ ID NO:27: Gly Gly Asn Tyr Leu Cys Arg Phe Gly Pro Gly Thr Trp Asp Cys Thr 67 1 5 10 Gly Phe Mrg Gly IN;FORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGT11 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28: Gly Glyv Asn Tyr Val Cys Mg Met Gly Pro Ile Thr Trp Ile Cys Thr 1 5 10 Pro Ala Gly Gly INFORMATION FOR SEQ ID NO:29: (i SEQUENCE CHARACTERISTICS: LENGT1 20 amino acids TYPE: amino acid' STRANDEDNESS- single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) -SEQUENCE DESCRIPTION:. SEQ ID NO:29: Gly Gly Lys Asp Val Cys Mrg Met Gly Pro ne Thr Trp Asp Cys Mrg 1 5 10 Ser Thr Gly Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERSTICS: IENGThS 23 amino acids (B TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 68 (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Gly Ser Tyr Leu Cys Arg Met Gly Pro Thr Thr Trp Leu Cys Thr 1 .5 10 Mla Gin Mrg Gly Gly Gly Asn INFORMATION FOR SEQ MD NO:3 1: SEQUENCE CHARACTERISTICS: LENGTR- 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 1: Gly Gly Asn Ty Lu Cys Mrg Met Gly Pro Ala Thr Trp Val Cys Gly 1 5 10 Mrg Met Gly Gly INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ* ID NO:32: Gly Gly Gin Tyr Lys Cys Arg Met Gly Pro Len Thr Trp Val Cys Gin 1 5 10 Tyr Ala Gly Gly INFORMATION FOR SEQ ID NO:33: -SEQUENCE CHARACTERISTICS: LENGTH- 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 69 (ii) MOLECULE TYPE: peptide' (xi) SEQUENCE DESCRIPTION: SEQ ID) NO:33: Gly Gly Asp Tyr Thr Cys Arg Met Gly Pro Met Thr Tip Ile Cys Thr 1 5 10 Ala Thr Arg Gly INFORMATION FOR SEQ I D NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino aicds TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear.

(ii) MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: SEQ ID NO:34:.

Gly Gly Val Tyr VlCys Arg Met Gly Pro Leu Thr Trp Giu Cys Thr 1 5 10 *1 Ala Scr Gly Gly INFORMATION FOR SEQ MD SEQUENCE CHARACTERISTICS: LENGTH1 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide SEQUENCE DESCRIPTON: SEQ ID Gly Gly Glu Tyr Ser Cys Arg Met Gly Pro Met Thr Tip Val Cys 1 5 10 Pro Thr Gly Gly 70 INFORMATION FOR SEQ ID NO:'6W SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (2d) SEQUENCE DESCRIPTIO N: SEQ ID NO:36: Gly Gly Glu Tyr Leu Cys Mrg Met Gly Pro Ble Thr Trp Val Cys Glu 1 5 10 Arg Tyr Gly Gly INFORMATION FOR SEQID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 20 a mino acids TYPE:. amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ BD NO:37: Gly Gly Asn Tyr Ile Cys Arg Met Gly Pro Met Thr Trp Val Cys Thr 1 5 10 Ala His Gly Gly INFORMATION FOR SEQ ID) NO:38: Qi) SEQUEN CE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUTENCE DESCRIPTION: SEQ ID NO:38: Gly Gly Asp Tyr Leu Cys Mrg Met Gly Pro Ala Thr Trp Val Cys Gly 1 5 10 Mrg Met Gly Gly 71 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino -acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ IID NO:3 9: Gly Gly Leu Tyr Leu Cys Arg Phe Gly Pro Val Thr Trp Asp, Cys Gly 1 5 10 Tyr Lys Gly Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH. 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRTION: SEQ ID Gly Gly Leu Tyr Ser Cys Mrg Met Gly Pro fle Thr Trp Val Cys Thr .1 5 10 Lys Ala Gly Gly INFORMATION FOR SEQ ID NO:41: SE~QUENCE CHARACTERISTICS: LENGTH:-20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE- peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Gly Gly Gly Tyr Hfis Cys Arg Met Gly Pro Met Thr Trp Val Cys Arg 72 1 5 10 Pro Val Gly Gly WNORMATION FOR SEQ IID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUTENCE DESCRIPTON: SEQ ID NO:42: Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 Pro Gin Gly Gly INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid (C).STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE T"YPE: peptide (0i) SEQUENCE DESCRIPTON: SEQ MD N0:43: Gly Gly Ile Tyr Lys Cys Len Met Gly Pro Leu Thr Trp Val Cys Thr 1 5 10 Pro Asp. Gly Gly INFORMATION FOR SEQ IID NO:44: SEQUENCE CHARAc]7ERIsTIC: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single 73 TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xid) SEQUENCE DESCRIPTION: SEQ ID NO:44: Gly Gly Leu Tyr Ser Cys ILeu Met Gly Pro Ile Thr Trp Leu Cys Lys 1 5 10 Pro Lys Gly Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS:' LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single (D).TOPOLOGY: linear (Hi) MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: SEQ ID Gly Gly Asp Tyr Hfis, Cys Arg Met Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 Pro Len Gly Gly INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Gly Gly Asp Tyr Ser Cys-Arg Met Gly Pro Thr Thr Trp Val Cys Thr 1 5 10 Pro Pro Gly Gly INFORMATION FOR SEQ ID NQ:47: SEQUENCE CHARACTERISTICS: 74 LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION.: SEQ MD NO:47: Gly Gly Asp Tyr Trp Cys Arg Met Gly Pro Ser ThrTrp Glu Cys Asn 1 5 10 Ala His Gly Gly INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: Gly Gly Lys Tyr Leu Cys Ser Phe Gly Pro Ile Thr Trp Val Cys Ala 1 5 10 Arg Tyr Gly Gly INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUJENCE DESCRIPTON: SEQ ID N0:49: Gly Gly Len, Tyr Lys Cys Mg Leu Gly Pro Ile Thr Trp Val Cys Ser 1 5 10. Pro Leu Gly Gly 75 INFORMATION FOR SEQ DD SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids T"YPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ Gly Gly Ser Tyr Thr Cys Arg Phe Gly Pro Glu Thr Trp Val Cys Arg 1 5 .10 Pro -Asn Gly Gly INFORMATION FOR SEQ ID NO:5 1: SEQUENCE CHARACTRISTCS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 1: Gly Gly Ser Tyr Ser Cys Mg Met Gly Pro le Thr Trp Val Cys Lys 1 5 10 Pro Gly Gly Gly INFORMATION FOR SEQ ID NO:52: Wi SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRLANDEDNESS: single AD TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTON: SEQ ID NO:52: 76 Gly Gly Ser Tyr Thr Cys Arg Met Gly Pro Hie Thr Trp Val Cys Leu 1 5. 10 Pro Ala Gly Gly INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: Gly Gly Leu Tyr Gin Cys Arg Met Gly Pro Met Thr Trp Val Cys Arg 1 5 10 Pro Gly Gly Gly INFORMATION FOR SEQ ID NO-54: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide.

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54: Gly Gly Asp 'Tyr Thr Cys Arg Met Gly Pro le Thr Trp Bie Cys Thr 1 5 10 Lys Ala Gly Gly INFORMATION FOR SEQ ID .(i).SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids (13) TYPE: amino acid' STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 77 (xi)-SEQUENCE DESCRPIMON: SEQ MD Gly Gly Val Tyr Ser Cys Mg Met Gly Pro Thr Thr Trp Gin Cys Asn 1. 5 10 Arg Tyr Val Gly- INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCPtPTION: SEQ ID NO:56: Gly Gly Ala Tyr Leu Cys His Met Gly Pro Ile Thr Trp Val Cys Mrg 1 5 10 Pro Gin Gly Gly INFORMATION FOR SEQ ID N0:57: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTON: SEQ ID) NO:57: Gly Gly Gin Tyr Ser Cys Mg Met Gly Pro Asn Thr Trp Val Cys Lys 1 5 10 Pro Val Gly Gly INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERSTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single 78 TOPOLOGY: linear (di) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTON: SEQ ID NO:58: Gly Gly Leu Ty Leu Cys Arg Met Gly Pro Va h Trp Glu Cys Gin 1 5 10 Pro Arg Gly Gly INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid ST.RANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTIN: 'SEQ ID NO:5'9: Gly Gly Leu Tyr Thr Cys Prm Met Gly Pro Ile Thr Trp Val Cys Len 1 5 10 Len Pro Gly Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTHI 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide (Ai) SEQUENCE DESCRIPTION: SEQ ID Gly Gly Leu Tyr Thr Cys Arg Met Gly Pro Val Thr Tip Val C)'s Thr 1 5 10 Gly Ala Gly Gly 79 INFORMATION FOR SEQ ID NO:6 1: SEQUENCE5 CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNBSS: Single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: Gly Gly- Val Tyr Lys Cys Arg Met Gly Pro Leu Thr Trp Glu Cys Arg.

1 5 10 Pro Thr rGly Gly TIFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (hi) MOLECULE TYPE: peptide (xd) SEQUENCE DESCRIPTION: SEQ ID NO:62: Gly Gly Asp Tyr Asn Cys Arg Phe Gly Pro Leu Thr Trp Val Cys Lys 1 5 10 Pro Ser Gly Gly INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide (xd) SEQUENCE DESCRIPTION: SEQ ID NO:63: Gly Gly Ser Tyr Lou Cys Arg Phe Gly Pro Thr Thr Trp Leu Cys Ser 1 5. 10 80 Ser Ala Gly Gly INFORMATION FOR SEQ ED NO:64: SEQUENCE CHARACTEPISTICS: LENGTH- 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: petide (xi) SEQUENCE DESCRPTON: SEQ ID NO:64: Gly Gly Ser Tyr Leu Cys Arg Met Gly Pro Thr Thr Trp Val Cys Thr 1 5 10 Arg Met Gly Gly INFORMATION FOR SEQ ID SEQUENCE CHA&RACTERISTICS: LENGT1 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY:* linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:65: Gly Gly Ser Tyr Leu Cys Arg Phe Gly Pro Thr Thr Trp Leu Cys Thr 1 5 10 Gin Arg Gly Gly INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERSTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 81 (Xi) SEQUENCE DESCRIPiJON. SEQ ID NO:66:.

Gly Gly Trp Val Thr Cys Arg MetGyPrleThTpVaCyGy 1 5 10 15 r leTrTpVa Y l Val is Gly Gly- WNORMATION FOR SEQ ID NO:67: SEQUENCE

CHARACTERISTCS:

LENGTH. 20 amino acids TYPE: amino acid STRANDEDNESS: Sin~gle TOPOLOGY: linear (ii) MO0LECUJLE TYPE: peptide (xi) SEQUENCE DESCRIPTION. SEQ ID NO:67:.

Gly Gly Gin Leu Leu Cys Gly Ie Gly Pro fle Thr Ti-p Val Cys Arg 1 5 10 Trp Val Gly Gly INFORMATION FOR SEQ ID NO:68: SEQUENCE

CHARACTERISTICS:

LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIMTON: SEQ ID N0:68: Gly Gly LYS Tyr Ser Cys Phe Met Gly Pro Thi- Thr Trp Val Cys Ser 1 5' 10 Pro Val Gly. Arg Gly -Val INFORMATION FOR SEQ ID NO:69: SEQUENCE

CHARACTERISTICS:

LENGTH:- 20 amino acids (13) TYPE: amino acid 82 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: -peptide (xi) SEQUENCE DESCRIPTON: SEQ ID NO:69: Gly Gly Trp Val Tyr Cys ArglIle Gly ProlIle Tr Trp Val Cys Asp 1 5 10 Thr Asn Gly Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE T YPE: peptide (Xi) SEQUENCE DESCRIPTON: SEQ MD Gly Gly Met Tyr Tyr Cys Mg Met Gly Pro Met Thr Trp Val Cys Lys 1 5 10 Gly Ala Gly Gly INFORMATION FOR SEQ D NO:7 1: SEQUENCE CHARACTEISTICS: LENGTH: 20 amino acids (B3) TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 00i MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTON: SEQ ID NO:71: Gly Gly Thr Thr GlnCys'TrplIle Gly Pro De Thr Trp Val Cys Arg.

.1 5 10 Ala Arg Gly Gly INFORMATION FOR SEQ ID NO:72: 83 SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear .0i) MOLECULE TYPE: peptide SEQUENCE DESCRIPTON: SEQ ID NO:72: *Gly Gly Pro Tyr His Cys Arg Met Gly ProlIle Thr Trp Val Cys Gly 1 5 10 Pro Val Gly Gly INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRA&NDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xid) SEQUENCE DESCRIPTION: SEQ ID NO:73: Gly Gly Glu Tyr Arg Cys Arg Met Gly Pro Ile Ser Tip Val Cys 1 5 10 Pro Gin Gly Gly INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: Gly Gly sn Tyr Thr Cys Arg Phe Gly Pro. Len Thr Tip Glu Cys Thr 1 5 10 -84- Pro Gin Gi, Gly Gly Ala flWORMATION FOR SEQ ID SEQUENCE aiARACTERISTICS: LENGTR* 20 amino acids (B TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 0ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRION:- SEQ, ID Gly Gly Ser Trp Asp CY.s Arg Ile Gly pro le. Thr Tn? Val Cys Lys 1 5. 10 1 .Trp Ser Gly Gly INFORMATION FOR- SEQ ID N0:76: SEQUENCE

CHARACT.ERISTICS:

LENGTH- 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY; linear (i)MOLECUIE TYPE: peptide (xi) SEQUENCE DES CRTON: S.EQ ID N0:76: Val Gly Asn TYr Met Cys His Phe Gly Pro Ie Thr Trp Val Cys Arg 10 Pro Gly Gly Gly WNORMATION FOR~ SEQ ID NO: 77: W i SEQUENCE CHARACTERiSTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRUPTON: SEQ ID N0:77: 85 Gly Gly Leu Tyr Leu Cys Arg Met Gly Pro Gin Thr Trp Met Cys Gln 1 5 10 Pro Gly Gly Gly INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: Gly Gly Asp Tyr Val Cys Arg Met Gly Pro Met Thr Trp Val Cys Ala 1 5 10 Pro Tyr Gly Arg INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: Gly Gly Trp Tyr Ser Cys Leu Met Gly Pro Met Thr Trp Val Cys Lys 1 5 10 Ala His Arg Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single CD) TOPOLOGY: linear 86 (ii) MOLECULE TYPE. peptide (xi) SEQUENCE DESCRIpnON: SEQ ID Gly Gly Lys Tyr Tyr Cys Trp Met Gly Pro Met Thr Trp Val Cys'Ser .1 5' 10 Pro Ala GIy Gly INFORMATION FOR SEQ ID NO:81: SEQUENCE

CHARACTERISTICS:

LENGTH. 20 amino acids TYPE: amino acid STRANDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: Gly Gly Tyr Val Met Cys Arg e GlyPro fle Thr Trp val Cys As 1 5 10 Ile Pro Gly Gly INFORMATION FOR SEQ ID NO:82:.

SEQUENCE

CHARACTERISTICS:

LENGTH. 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: Gly Ser Cys LenuGlCCys neG e Y pIle r Val CysArg 1 5 10 His Ala Gly Gly INFORMAUON FOR SEQ ID NO:83: 87 SEQUJENCE CHAkRACTERISTICS: LENGTH: 20 amino acids TYPE: amino, acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTON: SEQ"ID NO:83: Gly Gly Asn Tyr Phe Cys Arg Met GlyPro Ile Thr Trp Val Cys Gin 1 5 10 Arg Ser Val Gly INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNEFSS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ MD NO:84: Gly Gly Glu Tyr Ile Cys Arg Met Gly Pro Leu Thr Trp Glu Cys Lys 1 5 10 Arg Thr Gly Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY:.linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Gly Gly Leu Tyr Ala Cys Mrg Met Gly Pro le Thr Trp Val Cys Lys I1, 5 .10 Tyr Met Ala Gly 88 INFORM ATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid.

-STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ED NO: 86: Gly Gly Gin Tyr Leu Cys Thr Phe Gly Pro ie Thr Trp Len Cys Arg 1 5 10 Gly Ala Gly Gly INFORMATION FOR SEQ ID N0:87: SEQUENCE CHARACTERISTICS: LENGTH-- 20 amino acids TYPE: amino acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: Gly Gly Val TyT Ala Cys Arg Met Gly Pro Bie Thr Trp Val Cys Ser 1 5 10 Pro Leu Gly Gly INFORMATION FOR SEQ ID NO:88.: SEQUENCE CHARACTERISTICS: LENGTH, 20 amino acids TYPE: amino acid STRANDEDNES S: single TOPOLOGY: linear (ii) MOL ECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: Gly Gly Tyr Tr hr Cys Arg Met Gly Pro le Thr Trp Vai- Cys Ser 1 5 10 89 Ala His Gly Gly WNORMATION FOR SEQ ED NO:89: 0I) SEQUENCE

CRARACTERISTICS:

LENGTH 20 amino acids TYPE:. amino acid STRANDEDESS: single TOPOLOGY. linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRITION:- SEQ ID NO:89: Gly Gly Thr Tyr Lys Cys TrP Met Gly Pro met Thr Trp Val Cyg Arg 1 5 10 Pro Val Gly Gly WNORMATION FOR SEQ ID N0:90: SEQUENCE CHARACITflISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 0ii) MOLECULE~ TYPE: peptide SEQUENCE DESCRIPTION: SEQ ID "Gly Gly Asn Tyr Tyr Cys Arg P he Gly Pro lie Thr Phe Glu Cys FES 1 5 10 1 Pro Thr Gly Gly INORMATION FOR SEQ ID NO:91: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 amino acdds (B3) TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 90 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTON:. SEQ ID NO:9 1: Gly Gly Glu Tyr Leu Cys Arg Met Gly Pro Asn. Thr Trp Val Cys Thr 1 5 10 Pro Val Gly Gly INFORMATION FOR SEQ ID N0:91: SEQUENCE CHARACTISTICS: LENGTIL 20 amino acids TYPE: amino acid STRANDEDNBSS: single TOPOLOGY: linear (ii) MOLECULE TYPE:- peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: Gly Gly Leu Tyr Thr Cys Arg Met Gly Pro le Thr Trp Val Cys Leu 1 5 -10 Pro Ala Gly Gly INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS. single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: Gly Gly Leu Tyr Thr Cys Arg. Met Gly Pro le Thr Trp Val Cys Leu .1 5 10 Pro Ala Gly Gly 00000000000000000000000000 OODODODOD00000

Claims

1. A method of improving the bioactivity of a cell surface receptor comprising dimerizing a monomeric agonist of said cell surface receptor to form a dimer wherein each said monomeric agonist is a peptide of between 10 to 40 amino acids and contacting said dimer with said cell surface receptor to effect said improved biological activity wherein the agonist of said cell surface receptor is a GH agonist, PDGF agonist, EGF agonist, G-CSF agonist, TPO agonist, VEGF agonist, FGF agonist, insulin agonist, IL-3 agonist, IL-5 agonist, IL-6 agonist or IL-2 agonist.

2. A method of activating a cell surface receptor to induce biological activity of said cell surface receptor comprising dimerizing a monomeric agonist of said cell surface receptor to form a dimer wherein each said monomeric agonist is a peptide of between to 40 amino acids and contacting said dimer with said receptor thereby inducing said biological activity wherein the agonist of said cell surface receptor is a GH agonist, PDGF agonist, EGF agonist, G-CSF agonist, TPO agonist, VEGF agonist, FGF agonist, insulin agonist, IL-3 agonist, IL-5 agonist, IL-6 agonist or IL-2 agonist.

3. A method according to claim 1 or claim 2 wherein cell surface receptor is contacted with said dimer in vitro or in vivo.

4. A method of preparing a cell surface receptor agonist comprising dimerizing a cell surface receptor antagonist to form a dimer each said antagonist being a peptide of between 10 to 40 amino acids wherein said cell surface antagonist is a GH antagonist, PDGF antagonist, EGF antagonist, G-CSF antagonist, EGF antagonist, GM-CSF antagonist, TPO antagonist, VEGF antagonist, FGF antagonist, insulin antagonist, IL-3 antagonist, IL-5 antagonist, IL-6 antagonist, or an IL-2 antagonist.

A method of improving the bioactivity of a cell surface receptor substantially as herein described with reference to any one or more of the examples but excluding comparative examples.

6. A method of activating a cell surface receptor to induce biological activity of said cell surface receptor substantially as herein described with reference to any one or more of the examples but excluding comparative examples.

7. A method of preparing a cell surface receptor agonist substantially as herein described with reference to any one or more of the examples but excluding comparative examples.