WO2001017544A1 - Peptides that bind to urokinase receptor - Google Patents

Abstract

Adhesion and signaling by integrins requires their dynamic association with non-integrin membrane proteins. One such protein, the glycolipid-anchored urokinase receptor (uPAR), associates with and modifies the function of the β2 integrin Mac-1 (CD11b/CD18). We have identified a critical non-I domain binding site for uPAR on CD11b. Recombinant soluble uPAR and cells expressing uPAR bound to immobilized M25, binding being promoted by urokinase and blocked by soluble M25, but not a scrambled control or homologous peptides from other β2-associated a chains. In the a-propeller model of a chain folding, M25 spans an exposed loop on the ligand-binding, upper surfaced of αM, identifying uPAR as an atypical αM ligand. Although not blocking ligand binding to Mac-1, M25(25-100 νM) inhibited leukocyte adhesion to fibrinogen, vitronectin, ad cytokine-stimulated endothelial cells. M25 also blocked the association of uPAR with β1 integrins and impaired β1 integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen. Thus, uPAR associates with integrins directly and tat disruption of this association broadly impairs integrin function, providing a novel strategy for regulation of integrin function in the settings of inflammation and tumor progression.

Description

PEPTIDES THAT BIND TO UROKINASE RECEPTOR

TECHNICAL FIELD OF THE INVENTION This invention relates generally to methods and compositions for modulating or regulating cell adhesiveness The invention particularly relates to the use of newly identified binding sites on integrins for binding to urokinase receptors on cells

BACKGROUND OF THE INVENTION The integπn family of adhesion receptors is involved in inflammation, immunity, homeostasis, and tumor metastasis Integrins are heterodimenc proteins composed of an α and a β subunit Each subumt has a long 950-1 100 ammo acid extracellular domain and short

15-77 ammo acid cytoplasmic tail The adhesion of integπns to their ligands is dynamically regulated by conformational changes in integπn extracellular domains, by intracellular signal transduction pathways that reorganize lntracytoplasmic (especially cytoskeletal) connections, and by upregulation and redistπbution of integπns on the cell surface These dynamic aspects of mtegπn function are partly regulated by the interaction of integπns with neighboπng membrane-associated proteins, including the glycosyiphosphatidylmositol (GPI)-anchored urokinase receptor (uPAR, CD87)

Integπns interact with the urokmase receptor both physically and functionally

Physically, the urokmase receptor co-puπfies with the β- mtegπn Mac-1 (CD1 lb/CD 18) in monocvtes under mild denatuπng conditions The urokinase receptor and the Mac-1 integπn co-cluster in the presence of antibodies to either receptor and dissociate as cells polaπze in v in o in response to chemoattractants

The urokinase receptor also dynamically associates with β, (CD29) and β₃ (CD61) integπns Migration of tumor cells on vitronectin using α_vβ₃ (CD51/CD61) integπns requires the urokinase receptor Co-immunoprecipitation and puπfied recombmant proteins results indicate that the urokinase receptor forms complexes with a subset of β, and β₂ integπns (Wei et al , 273 Science 155 1-1555 (1996)) and modulates the signaling capacity of these integπns

(Wei et al . 144 J Cell Biol 1285-1294 (1999))

The functional consequences of integπn interactions with the urokinase receptor have been most extensively studied with β, mtegnns Sitπn et al , 97 J Chn Invest 1942-1951

(1996) showed that human monocytic cells rendered deficient in the urokinase receptor by treatment with antisense ohgonucleotides have defective Mac-1 -dependent adhesion to fibπnogen and migration on plastic A study of uPAR-deficient mice by May et al , 188 J Exp Med 1029-1037 (1998) reported defective β, integπn-dependent migration of neutrophils into the peritoneal cavity of mice injected with thioglycollate The Mac-1 integπn and the urokinase receptor form a bi-directional functional unit on monocytic cells, such that induction of the Mac-1 mtegπn and urokinase receptor expression on monocytic cell lines by cytokme stimulation confers urokinase and uPAR-dependent adhesion to vitronectin, which is further promoted by engagement of the Mac-1 integπn

By contrast, saturation of the urokinase receptor by exogenous urokinase inhibited Mac-1 function, implying the urokinase receptor can inhibit - as well as promote - integπn function (see Simon et al , 88 Blood 3185-3194 (1996)) Thus, a need remains in the art for a molecular explanation for this finding, as well as an identification of the sites of interaction between the Mac-1 integπn and the urokinase receptor, as these molecular events relate to diseases and physiological disorders

SUMMARY OF THE INVENTION

The invention is directed to polypeptides that bind to the urokinase receptor (uPAR) and inhibit interaction between the urokinase receptor and integπns, and methods of using these peptides The polypeptides coπespond to a non-I domain-binding site for the urokinase receptor present on β_^ integrins, and particularly on the Mac-1 integπn (at ammo acid residue 424-440 of the Mac-1 α_M mtegπn polypeptide. CDl lb) and on the α₃ integπn (VLA-3 alpha chain CD49c)

One such polypeptide (the "M25" polypeptide) inhibits leukocyte adhesion to fibπnogen, vitronectin, and cytokine-stimulated endothehal cells The M25 polypeptide also blocks the association between the urokinase receptor with β, integπns and impairs β, -dependent spreading and migration of vascular smooth muscle cells on fibronectm and collagen Another such polypeptide (the "A325" polypeptide) impairs β,-dependent spreading and migration of breast cancer cell line 231 cells

The polypeptides can disrupt the association of the urokinase receptor with a subset of β integπns and thereby block post-receptor integπn function Based on the ability of the polypeptides to inhibit cell adhesion, spreading, or migration, the polypeptides of the invention can be used to inhibit the interaction between integπn and the urokinase receptor for therapeutic regulation of mtegπn function in disorders such as inflammation and tumor - progression The methods are useful in the treatment of atherosclerosis, restenosis following percutaneous transluminal coronary angioplasty (PCTA), acute and chronic inflammatory disorders, and cancer

BRIEF DESCRIPTION OF THE DRAWINGS

FIG 1 is a schematic illustration of Mac-1 /p i 50,95 chimeras and subunit domain structure FIG 1 A shows 5 different chimeπc subunits compπsed of various fusions of the mtegπn polypeptides CDl lb (from Mac- 1 ) and CDl l c (from pl 50,95) that all retain the ligand binding I-domain of Mac- 1 FIG IB shows the N-termmal region (-450 ammo acids) of integπn α subunits, which has been proposed to fold into a β-propeller Insertion of the I domain between W2 and W3 is indicated in the Figures Peptide sequences of β, integπns spanning W4 align with the phage display-generated uPAR binding peptide 25 Homologous amino acid residues are indicated m bold type

FIG 2 is a bar graph showing that uPAR-expressing cells bind M25 Cytokine-stimulated THP-1 cells expressing uPAR were added to micotiter wells coated with M25, ScM25, L25 and P25 (80 μg/ml) and blocked with BSA The role of uPAR in cellular adhesion to M25 was examined by incubating cells with urokmase or receptor binding fragments thereof (ammo terminal fragments (ATF) of urokinase (10 nM)), polyclonal anti-uPAR antibody, or phosphoinositol-specfic phosphohpase After washing, adhesion was quantified by staining cells with Geimsa and measuπng absorbance at 540 nm Data shown are representative of three independent experiments

FIG 3 is a bar graph shows the effect of M25 on complex formation between uPAR and β, and β, integπns Biotinylated suPAR (100 nM) was added to wells coated with puπfied recombmant Mac- 1 in buffer containing 20 mM Hepes, 150 mM NaCl, 1 mM MnCl₂, pH 7 4, 0 05% Tween 20, and the plates were incubated for 90 min at 25°C in the presence and absence of M25 and ScM25 (25 pM) After washing, adhesion was quantified by the addition of avidin peroxidase Data are expressed as % control suPAR binding The binding of suPAR was inhibited by 25 μM M25 (% ιnhιbιtιon=57±6) but not by ScM25 Values represent mean ± SD, n=3 *Ctl vs M25 p<0 01 FIG 4 shows the effect of M25 on Mac- 1 -dependent ligand binding and adhesion FIG

4A is a chart showing that THP-1 cells were stimulated with PMA and adhesion to FGN-coated wells was assessed in the presence of 0-50 μM M25 (O), ScM25 (•), or 10 μg/ml anti-CDl lb mAb LPM19c (A). FIG. 4B is a bar graph showing that Mac-1 -dependent FGN binding was assessed by adding 1 μM ^l23l-FGN to PMA-stimulated adherent THP- 1 cells in the presence or absence of indicated peptides (25 μM). Data is expressed as LPM19c-inhibitable FGN binding (cpm). FIG. 4C is a bar graph showing the effect of M25 or control peptides (50 μM) on the adhesion of thioglycolate-elicited muπne neutrophils to TNF-α-stimulated endothelial cells. The dependence of this adhesion on Mac-1 was examined by incubating cells with the anti-CDl lb mAb Ml/70 (10 μg/ml). Data is expressed as % adhesion in the absence of peptides or Ml/70. Values represent mean ± SD, n=3.

FIG. 5 is a graph showing the effect of M25 on β-dependent smooth muscle cell (SMC) migration. Haptotactic migration of smooth muscle cells (SMC) toward fibronectin and collagen was investigated by seeding 10⁵ human saphenous vein smooth muscle cells into Transwell inserts precoated on the bottom with fibronectin (10 μg/ml) or collagen type I (20 μg/ml). Cells were cultured overnight with or without 25-100 μM M25 (■ , fibronectin-coated; • collagen-coated wells ) or 100 μM scM25 (Q fibronectin-coated; O collagen-coated wells). Cells on both sides of the filter were detached and counted. Data is expressed as % inhibition by the peptides; mean ± SD, n=3.

FIG. 6. is a bar graph showing the effect of 25myc peptides (50 μM or 100 μM) on the adhesion of thioglycolate-elicited murine neutrophils to TNF-α-stimulated endothelial cells. FIG. 7 is a graph showing the effect of M25-derived "loop" peptides on uPAR- dependent 293 cell adhesion to vitronectin.

DETAILED DESCRIPTION OF THE INVENTION Introduction. Tumor cells and other cells in the body secrete the urokinase-type plasminogen activator (urokinase; uPA). The proteolytic action of urokinase is potentiated by binding to cell surface receptors, allowing the urokinase to activate plasminogen to plasmin (which is then bound to physically adjacent plasma membrane receptors) and to subsequently cleave basement membrane constituents. Internalization of urokinase requires two cell surface receptors, (a) the low density lipoprotein receptor-related protein α2-macroglobulin receptor; and (b) the urokinase-type plasminogen activator receptor (urokinase receptor; uPAR; r humans, CD87; SEQ ID NO: 10). The urokinase receptor (uPAR) facilitates extracellular matrix proteolysis by accelerating plasmin formation at the cell surface. Although the urokinase receptor is anchored to the cell surface by a glycosylphosphatidylinositol linkage, this receptor has the capacity to initiate signal transduction responses The urokinase receptor also regulates cellular adhesion and migration by binding directly to the matπx protein vitronectin and by regulating the activity of integπns Urokinase (uPA) and the urokinase receptor (uPAR) in thus are important (a) for cell migration and chemotaxis, (b) for inflammatory cell invasion, and (c) for the invasive phenotype of several types of cancers

The urokinase receptor is a single chain 283 ammo acid receptor having 3 homologous domains of approximately 90 ammo acids each The N-terminal domain 1 (ammo acids 1-92) plays a dominant role in ligand (uPA) binding As descπbed above, the urokinase receptor forms a functional linkage with members of the β,, β₇, and β₃ integπn families, which then serve as signaling partners The urokinase receptor may also associate with other proteins that have tyrosine kmase activity The urokinase receptor is expressed by T-cells, NK cells, monocvtes, and neutrophils as well as non-hematopoietic cells that include vascular endothelial cells, fibroblasts, smooth muscle cells, keratmocytes, placental trophoblasts, hepatocytes, and a wide vaπety of tumor cells (including breast, colon, and prostate carcinoma, melanoma) Among leukocytes, constitutive expression is weak or absent but exposure to soluble activating factors (e g , hpoplysacchaπde in monocytes, IL-2 in T- lymphocytes) induces heightened message and cell surface expression

As described above, integrins interact with the urokinase receptor both physically and functionally On the basis of homology with G protein-coupled receptors, Spπnger 94 Proc Natl Acad Sci USA 65-72 ( 1997)) has proposed that the N-terminal region (-450 ammo acids) of an integπn α subunit folds into a β-propeller In this model, (see FIG IB) repeating units (W1 -W7) of anti-parallel β-sheets connected by surface loops (-60 ammo acids/unit) aπange into a torus around a small central cavity The upper surface loops contain the major hgand-bmdmg sites that synergize with the RGD-bindmg site on the β chain to define the specificity and affinity of interactions of integπns with their ligands The disc-like propeller is connected to and spans the plasma membrane by a C-terminal stalk In β₂ integπns, a 200 ammo acid I-domam, also directly implicated m ligand binding, is inserted between repeats 2 and 3 (Diamond et al , 120 J Cell Biol 1031 -1043 ( 1993), Michishita et al , 72 Cell 857-867 ( 1993)) We have identified a non-I domain peptide sequence, M25

(PRYQHIGLVAMFRQNTG, SEQ ID NO 1 ), within the α chain of the Mac-1 integπn (α_M 424-440, see SEQ ID NO 1 1 ) that critically regulates the binding of Mac-1 to the urokinase receptor (uPAR) In the β-propeller model, M25 comprises the entire upper loop sequence of W4 and extends into the third β strand of this repeat The upper W4 loops of other α integrm chains (cf, LFA-1 (SEQ ID NO 12) and pi 50,95 (SEQ ID NO 13)) had been implicated by Iπe et al . 94 Proc Natl Acad Sci USA 7198-7203 (1997) in integπn ligand binding By contrast, our results indicate that the urokinase receptor, which does not contain an RGD sequence, is an atypical integπn ligand

As a non-I domain binding ligand that influences the function of the Mac-1 integπn, the urokinase receptor resembles factor X (Zhou et al , 269 J Biol Chem 17075-17079 (1994), Mesπ et α/ , 273 J Biol Chem 744-748 (1998)) The precise region within the Mac-1 integπn mediating factor X binding is, however, unknown Preliminary expeπments indicate that factor X fails to compete with soluble urokinase receptor (suPAR) binding to immobilized M25

Identification of a uPAR-bindmg sequence in α and the recognition that the urokinase receptor is a cis-actmg integπn ligand may explain several previously obscure features of the interaction between the Mac-1 integπn and the urokinase receptor

(a) the strong promoting effect of the "active" state of the Mac-1 integπn on uPAR/Mac-1 binding, as reported by Wei et al , 273 Science 1551-1555 (1996)),

(b) the ability of suPAR to block the binding of known ligands such as factor X and fibrmogen (FGN) to the Mac-1 integπn, as reported by Simon et al , 88 Blood 3185-3194 (1996)), and

(c) the co-localization of the urokinase receptor and the Mac-1 integπn on leukocytes expressing these receptors (Bohuslav et al 181 J Exp Med 1381-1390 (1995), Xue et al , 152 J Immunol 4630-4640 (1994))

(d) In addition, the positioning of M25 on the Mac-1 integπn may also explain our observation that disruption of uPAR/Mac-1 association on PMA-stimulated THP-1 cells by the M25 polypeptide promotes fibπnogen binding to these cells (see EXAMPLE 1) The I domain-binding site for fibπnogen and the W4 binding site for the urokinase receptor may be in close proximity in the folded Mac-1

(e) If so, the additional finding the urokinase promotes the binding of the urokinase receptor to the Mac-1 integπn, as reflected in M25 binding (FIG 2B), may explain why saturation of the urokinase receptor with urokinase inhibits fibπnogen binding/turnover by Mac-1 (Simon et al , 88 Blood 3185-3194 (1996)) (see TABLE 3) (f) Our observations may also explain the suggestion of May et al , 188 J Exp Med 1029-1037 (1998)) that the urokinase receptor may "activate" integπns Ligand binding to integrins is a recognized mechanism for integπn activation The intπguing possibility that the urokinase receptor may activate the Mac-1 integπn by trans-type intercellular interactions as well as through the -type interactions identified here remains to be explored

Polvpepttcles of the invention The polypeptide of the invention is a ligand for the urokmase receptor (uPAR) A "ligand" is a molecule that contains a structural portion that is bound by specific interaction with a particular receptor molecule, such as the urokinase receptor, an integπn, or an antibody The polypeptide of the invention can be a polypeptide having the sequence for the M25 polypeptide The specificity of the interaction between the M25 polypeptide and the urokinase receptor was established using peptides corresponding to analogous sequences from LFA-1 (SEQ ID NO 2, "L25") or pi 50,95 (SEQ ID NO 3, "P25") Biotinylated suPAR bound immobilized M25 and this binding was blocked efficiently by soluble M25 polypeptide but not by the ScM25 polypeptide (HQIPGAYRGVNQRFTML, SEQ ID NO 5), L25, or P25 polypeptides These results indicate that among β₂ mtegπns, the urokinase receptor appears to react preferentially with Mac-1 in this otherwise homologous (>80%) a subunit region between the I-domain and highly conserved divalent cation repeats

The polypeptide of the invention can also be a polypeptide having the sequence for the human α₃ peptide (PRHRHMGAVFLLSQEAG, "A325", SEQ ID NO 4), from the predicted strand 2 W4 loop of the α3 integπn Like the M25 polypeptide, the A325 polypeptide is active in inhibiting the binding of the urokinase receptor to β mtegnn

More generally, the polypeptide of the invention has identifying structural characteπstics, being substantially homologous to the M25 polypeptide "Substantially homologous" means that a particular polypeptide sequence vanes from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilaπty between the reference sequence (M25) and the subject sequences For purposes of the invention, polypeptides having greater than 40% similaπty, equivalent biological activity, and equivalent expression characteπstics are considered substantially homologous to the M25 polypeptide For purposes of determining homology or similaπty, truncation or internal deletions of the reference sequence should be disregarded, as should subsequent modifications of the molecule, e g , glycosylation Sequences having lesser degrees of homology and comparable bioactivity are considered "equivalents"

When a polypeptide of the invention has a sequence that is not identical to the sequence of the M25 polypeptide, one or more conservative or non-conservative substitutions have typically been made Examples of conservative substitutions are provided by the BLAST computer program, the National Center for Biotechnology Information (NIH) sequence similarity search tool designed to support analysis of nucleotide and protein databases and other programs such as FASTA and Smith- Waterman Access to the methods and databases for sequence alignment has been simplified with the development of programs such as the Biology Workbench (available from the University of Illinois) Sequence alignments generally are based on the dynamic programming algoπthm of Needleman & Wunsch, J Mol Biol 48 442-453 (1970) Substitution schemes for scoπng alignments are known in the art (see, Dayoff e/ α/ , 91 Meth Enzymol 524-545 (1983), Hemkoff & Hemkoff, 89 Proc Natl Acad Sci USA 10915- 10919 (1992), Johnson & Oveπngton, 233 J Mol Biol 716-738 (1993), and Pearson, Protein Sci 4 1145-1160 (1995))

In one embodiment, the polypeptide of the invention has a consensus sequence as provided below in TABLE 1

TABLE 1

Polypeptide Ammo Acid Sequence

M25, SEQ ID NO 1 PRYQHI GLVAMFRQNTG α₃ sequence. SEQ ID NO 4 PRHRHMGAVFLLSQEAG

First consensus PRXXHXGXVXXXXQXXG

In this first consensus sequence (PRXXHXGXVXXXXQXXG , SEQ ID NO 6), X can be any ammo acid, such that X represents an ammo acid residue or a deletion The polypeptide of the invention can be deπved from any homologous W4 loop of another α integπn polypeptide, m which the polypeptide functions like the M25 polypeptide and the α₃ polypeptide The polypeptide can also be a fusion proteins compπsed of the peptide and other peptides (see EXAMPLE 6) and is thus useful for constructing a therapeutic, such as a signal peptide or a C-terminal tag (m\ c or His tag) These fusion proteins can also be used in gene transfer type therapeutics Other consensus sequences can be obtained, for example, by compaπson of the peptide M25 (which effectively binds to the urokinase receptor) with the peptides L25 and P25, as provided below in TABLE 2.

TABLE 2

Polypeptide Amino Acid Sequence

M25; SEQ ID NO: l PRYQHI GLVAMFRQNTG

L25; SEQ ID NO:2 PRYQHMGRVLLFQEPQG

P25; SEQ ID NO:3 PRYQHTGKAVI FTQVSR

Second consensus PRYQHXGXXXXFXXXXX

In this second consensus sequence (PRYQHXGXXXXFXXXXX; SEQ ID NO:7), X can be any amino acid or a deletion. Polypeptides having the second consensus sequence can readily tested for activity using a biochemical urokinase receptor binding assay or in vitro activity assay that tests for inhibition of binding between the urokinase receptor and integrm or inhibition of cellular binding or migration, such as are provided in the EXAMPLES below. Thus, the determination of which polypeptides having the second consensus sequence are active can be determined without undue experimentation.

The polypeptide of the invention may be of any size so long as the urokinase receptor-binding activity is retained. In particular, the polypeptide can be of any size from 6 to about 50 amino acids in length. Thus, the size of the polypeptide can be from 7, 8, 49, or 50 amino acids in length, or of any size in between. General methods for the construction and use of peptides involved in integrin-mediated binding reactions are provided in United States patent 5,939,525; 5,932,421 ; 5,912,234; and 5,843,774, all of which are incorporated by reference.

The polypeptide of the invention inhibits binding between the urokinase receptor and several classes of antigens. The urokinase receptor interacts not only with the Mac-1 integrm, but also with a class of β, integrins. The rules of physical interaction between the urokinase receptor and β, integrins appear to be similar to that observed for the Mac-1 integrm. Binding of suPAR to α,β, is promoted by activating TS2/16 antibodies and matrix engagement (Xue et al, 57 Cancer Res 1682-1689 (1997)) and suPAR blocks fibronectin binding to activated α₅βl (Wei et al, 273 Science 155 1-1555 (1996)). The fact that M25 blocks the co-precipitation of the urokinase receptor with β, integrins (see, EXAMPLE 3) suggests a similar mode of physical interaction. Numerous studies document that the urokinase receptor promotes normal β, and β₂ integπn functions, including Mac-1 -dependent adhesion to fibπnogen and endothelial cells in vitro (Sitπn et al , 97 J Clm Invest 1942-1951 (1996), Simon et al , 88 Blood 3185-3194 (1996)) and vascular emigration vivo (May et al , 188 J Exp Med 1029-1037 (1998)) That M25 impairs Mac-1 dependent adhesion to fibπnogen and stimulated endothelial cells (see, EXAMPLE 4) and β, -dependent migration of smooth muscle cells on fibronectin and collagen (see EXAMPLE 5) is consistent with these pπor reports How can this occur⁹ Two observations reported here are instructive First, M25 impairs Mac-1 dependent adhesion of THP-1 cells to fibπnogen while at the same time promoting bmding of soluble fibπnogen to the Mac-1 integπn on these cells (see, EXAMPLE 1) Second, disruption of uPAR/integπn complexes by uPAR-bmding peptides not only removes the urokinase receptor from integπn complexes but also Src-family kmases (FIG 5) Whereas soluble ligands can bind to surface integπns at 4°C, adhesion of cells to immobilized ligands requires both integπn clusteπng and cell signaling, events collectively termed "post-receptor binding events" (Wei et al , 144 J Cell Biol 1285-1294 (1999)) Because the urokinase receptor promotes the association of signaling molecules with integπns, the pπmary role of urokinase receptors in integnn clusters is to promote the post- receptor binding events important to β, and β₂ integπn function Thus, the ligand-hke binding of the urokinase receptor to the Mac-1 integπn is a mechanism to enrich and localize integπn clusters with important signaling molecules necessary for efficient integπn signaling, even though this may be at the expense of decreasing the total number of ligand binding sites available For example, the number of fibπnogen binding sites on THP-1 cells is lower when the urokinase receptor is associated with the Mac-1 integπn and yet THP-1 cells only adhere well to fibrmogen when the urokinase receptor is Mac-1 -associated (see, EXAMPLE 1) The available data are thus consistent with urokinase receptor regulating post-receptor binding events important to β, integπn function (FIG 5)

Likewise, the A325 polypeptide inhibits binding between urokinase and integπns (see EXAMPLE 7)

Substitutions, modifications, analogs, and fragments The polypeptide of the invention need not be identical to the ammo acid residue sequence of the M25 polypeptide, for example, so long as the polypeptides are able to bind to the urokinase receptor The polypeptide of the invention also includes any analog, fragment or chemical deπvative of the M25 polypeptide, so long as the polypeptide is functionally active The term "analog" includes a polypeptide havmg an amino acid residue sequence substantially identical to the M25 polypeptide sequence in which one or more amino acid residues have been conservatively substituted with a functionally similar residue (for example the L25 polypeptide and the P25 polypeptide) Peptide equivalents can differ from the M25 polypeptide polypeptides by the substitution or modification of side chains or functional groups, without destroying biological function Modifications can include, for example, additions, deletions, or substitutions of amino acids residues (see, FIG IB), substitutions with compounds that mimic amino acid structure or functions, and the addition of chemical moieties, such as amino or acetyl groups An "ammo acid residue" is an ammo acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages A "fragment" is a polypeptide of the invention having an amino acid residue sequence shorter than 50 amino acids (see EXAMPLE 6)

The amino acid residues described herein are preferably in the "L" isomeπc form However, residues in the "D" isomeπc form can be substituted for any L-ammo acid residue, as long as the polypeptides inhibit interaction between the urokinase receptor and integπns This definition includes, unless otherwise specifically indicated, chemically modified amino acids, including amino acid analogs (such as penicillamine, 3-mercapto-D-valme), naturally occurring non-proteogemc amino acids (such as norleucme), and chemically synthesized compounds that have properties known in the art to be characteπstic of an ammo acid The term "proteogemc" indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways

The term "conservative substitution" also includes the use of a chemically deπvatized residue in place of a non-deπvatized residue provided that such polypeptide displays the requisite activity "Chemical deπvative" refers to a polypeptide of the invention having one or more residues chemically deπvatized by reaction of a functional side group Such deπvatized molecules include for example, those molecules m which free amino groups have been deπvatized to form amine hydrochloπdes, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups Free carboxyl groups may be deπvatized to form salts, methyl and ethyl esters or other types of esters or hydrazides Free hydroxyl groups may be deπvatized to form O-acyl or O-alkyl deπvatives Also included as chemical deπvatives are those peptides that contain one or more naturally occurπng ammo acid deπvatives of the twenty standard amino acids For examples, 4-hydroxyprohne may be substituted for prohne, 5-hydroxylysιne may be substituted for lysine, 3-methylhιstιdme may be substituted for histidme, homoseπne may be substituted for seπne, and ormfhine may be substituted for lysine

Some useful modifications are designed to increase the stability of the polypeptide in solution, and therefore serve to prolong half life of the polypeptides in solutions, particularly biological fluids such as blood, plasma or serum, by blocking proteolytic activity in the blood A polypeptide can have a stabilizing group at one or both termini Typical stabilizing groups include amido, acetyl, benzyl, phenyl, tosyl, alkoxycarbonyl, alkyl carbonyl, benzyloxycarbonyl and similar end group modifications Additional modifications include using a "L" amino acid in place of a "D" ammo acid at the termini, cychzation of the polypeptide, and amide rather than ammo or carboxy termini to inhibit exopeptidase activity A polypeptide of the invention may or may not be glycosylated The polypeptides are not glycosylated, for example, when are produced directly by peptide synthesis techniques or are produced in a procaryotic cell transformed with a recombmant polynucleotide of the invention Eucaryotically-produced peptide molecules are typically glycosylated Binding The term "binding" refers to the ability of a polypeptide to interact with the urokinase receptor or an integπn such that the interaction between the peptide and integπn is relatively specific The term "relatively specific" means that the affinity of bmding of the integπn and peptide is about 1x10⁵ M or less Therefore, the term "binding" does not encompass non-specific binding, such as non-specific adsorption to a surface Non-specific binding can be readily identified by including the appropπate controls in a binding assay (see EXAMPLE 3, use of the Sc25 polypeptide)

A characteπstic of such bmding is that the bound peptide can be detached or prevented from binding to an integπn by initial contact with, or specific elution with, the polypeptide of the invention In addition, specific binding can be disrupted using an agent such as EDTA, which renders an integπn inactive, or by a nonspecific protein denaturant such as low pH buffer

Using the binding assays descπbed herein and other binding assays well known in the art, the relative binding affinity of a peptide for an integπn can be determined The term "relative binding affinity" refers to the ability of one peptide, in comparison to a second peptide, to inhibit the binding to the urokinase receptor The relative binding affinity of a peptide is determined by competitive binding assays, which identify peptide motifs that can inhibit the binding of a peptide to an integπn The relative binding affinities of vaπous peptides of the invention can be compared by determining the half-maximal inhibitory concentration (IC.,₀) of these peptides Typically, peptides are compared with the standard integπn-binding peptide Competitive binding assays using peptides displayed on phage obtained from phage display hbraπes, such as the phage display library descπbed in Scott & Smith, 249 Science 1738-1740 (1990) can also be employed to determine relative binding affinities

The term "high binding affinity" refers to peptides that have an IC₅₀ of 1x10⁷ M or less m a competitive binding assay for integπn Peptides having a high binding affinity are characteπzed by the ability to bind integπns immobilized on a surface at very low concentrations such as about 10 ng/well

The term "selectively binding" or "selectively binds" refers to a peptide that has a high binding affinity, exclusively or predominately, to a designated integπn Such selective binding can be determined, for example, by inhibition of binding of urokinase receptor to a particular integπn, such as the Mac-1 integπn, but not to other integπns How to make the polypeptide of the invention The polypeptides of the invention can be produced by well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods, as descπbed by Dugas & Penney, Bworganic Chemistry, 54- 92 (Spπnger-Verlag, New York, 1981) Alternatively, a polypeptide of the invention can be synthesized by using well known methods including recombmant methods and chemical synthesis

A polypeptide of the invention can be chemically synthesized, for example, by the solid phase peptide synthesis of Merπfield et al , 85 J Am Chem. Soc 2149 (1964) Alternatively, a polypeptide of the invention can be synthesized using standard solution methods (see, for example, Bodanszky, Principles of Peptide Synthesis (Spπnger-Verlag, 1984)) Newly synthesized peptides can be puπfied, for example, by high performance liquid chromatography (HPLC), and can be characteπzed using, for example, mass spectrometry or amino acid sequence analysis

The polypeptides of the invention are particularly useful when they are maintained in a constrained secondary conformation The terms "constrained secondary structure," "stabilized" and "conformationally stabilized" indicate that the peptide bonds compπsing the peptide are not able to rotate freely but instead are maintained in a relatively fixed structure A method for constraining the secondary structure of a newly synthesized linear peptide is to cychze the peptide using any of vaπous methods well known in the art For example, a cychzed polypeptide of the invention can be prepared by forming a peptide bond between non-adjacent amino acid residues as descπbed, for example, by Schiller et al , 25 Int J Pent Prot Res 171 (1985) Peptides can be synthesized on the Merπfield resm by assembling the linear peptide chain using N -Fmoc-amιno acids and Boc and tertiary-butyl proteins Following the release of the peptide from the resm, a peptide bond can be formed between the ammo and carboxy termini

A newly synthesized linear peptide can also be cychzed by the formation of a bond between reactive ammo acid side chains For example, a peptide containing a cysteme-pair can be synthesized and a disulfide bπdge can be formed by oxidizing a dilute aqueous solution of the peptide with K₃Fe(CN)₆ Alternatively, a lactam such as an ,-((-glutamyl)-lysme bond can be formed between lysine and glutamic acid residues, a lysmonorleucine bond can be formed between lysine and leucine residues or a dityrosine bond can be formed between two tyrosme residues Cyclic peptides can be constructed to contain, for example, four lysine residues, which can form the heterocyclic structure of desmosine (see, for example, Devlin, Textbook of Biochemistry 3rd Edition (1992) Methods for forming these and other bonds are well known in the art and are based on well known rules of chemical reactivity (Morπson & Boyd, Organic Chemistry 6th Edition (Prentice Hall, 1992)) Recombmant methods of producing a polypeptide of the invention through the introduction of a vector including a polypeptide encoding the peptide into a suitable host cell is well known in the art, such as is descπbed in Sambrook et al , Molecular Cloning A Laboratory Manual, 2^nd Edition (Cold Spπng Harbor Press, 1989) The term "recombmant" refers to the molecular biological technology for combining polynucleotides to produce useful biological products, and to the polynucleotides and peptides produced by this technology The polynucleotide can be a recombmant construct (such as a vector or plasmid) which contains the polynucleotide encoding the peptide or fusion protein under the operative control of polynucleotides encoding regulatory elements such as promoters, termination signals, and the like "Operatively linked" refers to a juxtaposition wherein the components so descπbed are m a relationship permitting them to function in their intended manner A control sequence operatively linked to a coding sequence is hgated such that expression of the coding sequence is achieved under conditions compatible with the control sequences "Control sequence" refers to polynucleotide sequences which are necessary to effect the expression of coding and non- coding sequences to which they are gated Control sequences generally include promoter, πbosomal binding site, and transcπption termination sequence Determinations of the sequences for the polynucleotide coding region that codes for the peptides descπbed herein can be performed using commercially available computer programs, such as DNA Stπder and Wisconsin GCG Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences can be constructed which encode the claimed peptides (see, Watson et al , Molecular Biology' of the Gene, 436-437 (The Benjamin Cummmgs Publishing Co , 1987)) In addition, "control sequences" refers to sequences which control the processing of the peptide encoded within the coding sequence, these can include, but are not limited to sequences controlling secretion, protease cleavage, and glycosylation of the peptide The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences A "coding sequence" is a polynucleotide sequence that is transcπbed and translated into a polypeptide Two coding polynucleotides are "operably linked" if the linkage results in a continuously translatable sequence without alteration or interruption of the tπplet reading frame A polynucleotide is operably linked to a gene expression element if the linkage results in the proper function of that gene expression element to result in expression of the coding sequence "Transformation" is the insertion of an exogenous polynucleotide (i e a "transgene") into a host cell The exogenous polynucleotide is integrated withm the host genome A polynucleotide is "capable of expressing" a peptide if it contains nucleotide sequences which contain transcπptional and translational regulatory information and such sequences are "operably linked" to polynucleotide which encode the peptide A polynucleotide that encodes a peptide coding region can be then amplified, for example, by preparation in a bacteπal or viral vector, according to conventional methods, for example, descπbed in Sambrook et al , Molecular Cloning A Laboratory' Manual 2^nd Edition (Cold Spπng Harbor Press, 1989) Expression vehicles include plasmids or other vectors The polynucleotide encoding the polypeptide of the invention can be prepared by chemical synthesis methods or by recombmant techniques The polypeptides can be prepared conventionally by chemical synthesis techniques, such as descπbed by Merπfield, 85 J Amer Chem Soc 2149-2154 (1963) (see, Stemmer et al, 164 Gene 49 (1995)) Synthetic genes, the in vitro or in vivo transcπption and translation of which will result in the production of the protein can be constructed by techniques well known in the art (see Brown et al , 68 Methods in Enzymology 109-151 (1979)) The coding polynucleotide can be generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (commercially available from Applied Biosystems, Inc , 850 Lincoln Center Dπve, Foster City, Calif 94404)

Alternatively, systems for cloning and expressing polypeptide of the invention include vaπous microorganisms and cells that are well known in recombmant technology These include, for example, vaπous strains of E coli, Bacillus, Streptomyces, and Saccharomvces, as well as mammalian, yeast and insect cells Suitable vectors are known and available from pπv ate and public laboratoπes and depositoπes and from commercial vendors See, Sambrook et al , Molecular Cloning A Laboratory Manual, 2^nd Edition (Cold Spπng Harbor Press, 1989) See, also PCT International patent application WO 94/01139) These vectors permit infection of patient's cells and expression of the synthetic gene sequence in vivo or expression of it as a peptide or fusion protein in vitro

Polynucleotide gene expression elements useful for the expression of cDNA encoding peptides include, but are not limited to (a) viral transcπption promoters and their enhancer elements, such as the SV40 early promoter, Rous sarcoma virus LTR, and Moloney muπne leukemia virus LTR, (b) splice regions and polyadenylation sites such as those deπved from the SV40 late region, and (c) polyadenylation sites such as m SV40 Recipient cells capable of expressing the gene product are then transfected The transfected recipient cells are cultured under conditions that permit expression of the gene products, which are recovered from the culture Host mammalian cells, such as Chinese Hamster ovary cells (CHO) or COS-1 cells, can be used These hosts can be used in connection with poxvirus vectors, such as vaccinia or swmepox Suitable non-pathogenic viruses that can be engineered to carry the synthetic gene into the cells of the host include poxviruses, such as vaccinia, adenovirus, retroviruses and the like A number of such non-pathogenic viruses are commonly used for human gene therapy, and as earner for other vaccine agents, and are known and selectable by one of skill m the art The selection of other suitable host cells and methods for transformation, culture, amplification, screening and product production and puπfication can be performed by one of skill m the art by reference to known techniques (see e g Gethmg & Sambrook, 293 Nature 620-625 (1981))

Another preferred system includes pSecTag2 expression vectors (Invitrogen, San Diego. CA) These 5 2 kb mammalian expression vectors offer features for secretion, punfication, and detection of fusion proteins The vectors carry the secretion signal from the V-J2-C region of the mouse Ig 6-cham for efficient secretion of recombmant proteins from a vaπety of mammalian cell lines The vectors also carry a C-termmal tag sequence that allows rapid punfication and detection of fusion proteins Recombmant proteins are fused to six histidine residues to allow one-step punfication (which can be removed following punfication) and the anti-mvc epitope for rapid detection The M25 polypeptide has been expressed in this system, to generate the 25myc polypeptide (STYHHLSLGYMYYLNGGEQKLISEEDL, see EXAMPLE 6)

When produced by conventional recombmant means, peptide can be isolated either from the cellular contents by conventional lysis techniques or from cell medium by conventional methods, such as chromatography (see, e g , Sambrook et al , Molecular

Cloning A Laboratory Manual, 2^nd Edition (Cold Spπng Harbor Press, 1989) Methods for recovenng an expressed recombmant polypeptide are well known in the art and include fractionation of the protein-containmg portion of the culture using well known biochemical techniques For instance, the methods of gel filtration, gel chromatography, ultrafiltration, electrophoresis, ion exchange, affinity chromatography and the like, such as are known for protein fractionations, can be used to isolate the expressed proteins found in the culture In addition, immunochemical methods, such as lmmunoaffmity, immunoadsorption and the like can be performed using well known methods

Method of treating using the polypeptide of the invention Currently, strategies to inhibit integπn function m settings of deleteπous inflammation and tumor progression are based on direct inhibition of integπn ligand binding to matnx proteins (such as fibronectin) or cellular counter receptors (such as Ig-family of adhesion receptors) The M25 polypeptide provided here is the first polypeptide in the extracellular domain of an integπn that broadly impairs integπn adhesion and migration to matnx proteins without directly inhibiting overall ligand binding

Inhibition of mtegπn/uPAR interactions could complement existing strategies for therapeutic regulation of integπn function A particularly attractive feature of this strategy is that the urokinase receptor is not constitutively expressed on most cells, or expressed only at low levels Instead, the urokinase receptor is strongly upregulated on migrating inflammatory cells and on tumor cells, where the urokinase receptor expression strongly coπelates with tumor progression (see, Hofmann et al , 78 Cancer 487-492 (1996)) Thus, reagents such as M25 may have the benefit that they should only impair mtegnn function of the cells most desirable to target

The invention is useful for modulating cellular migration, particularly with disorders involving leukocyte adhesion and migration, such as acute and chronic inflammatory disease, restenosis following percutaneous transluminal coronary angioplasty (PCTA) (particularly as it relates to the proliferation of smooth muscle cells), and transplant rejection Also included are disorders involving endothelial cell migration Further included are methods for inhibiting angiogenesis, coronary and artery bypass procedures, and the treatment of obesity

The invention is useful for preventing, inhibiting, or ameliorating inflammatory and immune reactions associated with vaπous injury and disease conditions The term "subject" means any mammal to which the compositions of the invention may be administered Subjects specifically intended for treatment with the compositions and methodologies of the invention include humans

Inflammation is the body's reaction to injury and infection Three major events are involved in inflammation (1) increased blood supply to the injured or infected area, (2) increased capillary permeability enabled by retraction of endothelial cells, and (3) migration of leukocytes out of the capillaπes and into the smroundmg tissue (hereinafter referred to as cellular infiltration, see Roitt et al Immunology, (Grower Medical Publishing, New York, 1989) Inflammatory cells and mediators including leukocytes, cytokines, oxygen radicals, complement and arachidonate metabolite damage capillary endothehum and allow fluid and protein to leak across capillaπes Inflammation also involves the release of inflammatory mediators (inflammatory cytokines, platelet activating factor, complement, leukotπenes, oxygen metabolites, and the like) Inflammatory responses are thus induced both at the ongmal site of trauma and also in the vasculature and remote vasculaπzed sites Neutrophils, eosinophils, mast cells, lymphocytes and macrophages contnbute to the inflammatory response Neutrophils contnbute by generating oxidants or releasing proteases that damage the microvasculature or adjacent tissue Cell death and tissue damage due to complement and inflammatory cell mechanisms lead to organ failure or decreased organ function The activation of mediators by a local injury can also cause a remote injury to highly vasculanzed organs

Among the disease and injury conditions that benefit from the compositions and methodologies of the invention are inflammatory pathologies such as asthma, systemic lupus erythematosus, vascuhtis, inflammatory autoimmune myositis, autoimmune thyroiditis, multiple sclerosis and arthritis There are many different types of arthntis clinically recognized, the most common being rheumatoid arthntis However, the inflammatory pathway relevant to the pathogenesis of rheumatoid arthntis is also likely relevant to the pathogenesis of other types of arthntis e g osteo, psoπatic and spondyloarthropathies, since the synovial pathologies m all these forms of arthntis is in many cases, the same Psonasis is also treatable by the compositions and methodologies of the invention, as is caused by an autoimmune inflammatory response to a set of antigens in the skin The invention is also directed to treatment of systemic shock and the many resultant clinical conditions associated with systemic shock The compositions and methodologies of the invention are useful in the treatment of ischemia, reperfusion injury, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), adult respiratory distress syndrome (ARDS), systemic lupus erythematosus (SLE), myocardial dysfunction, vasodilation, hypotension, endothelial injury, disseminated intravascular coagulation, and liver, kidney and central nervous system (CNS) failure The invention is useful for inhibiting tumor progression In the metastasis of the cancer cells, cancer cells are first released from a cancer-developed site, and are earned and moved through a human body with blood The free cancer cells are adhered to the intravascular endothelial cells through the rolling phenomenon Then, the cancer cells pass between the intravascular endothelial cells, and enter vascular tissues, so that a new cancer cell nest is formed Administration of the polypeptide of the invention can usefully inhibit the migration of metastatic cells For example, administration of the polypeptide of the invention can inhibit binding of cells to laminm, including lammin 5 Lamιmn-5 is a marker of invading cancer cells in some human carcinomas and is co-expressed with the urokinase receptor m budding cancer cells in colon adenocarcmomas and squamous cell carcinomas (i e cells of epithelial oπgin, see Pyke et al , (18) Cancer Res 554132-9 ( 1995)) The co-localization of lammm-5 and urokinase receptor in a subset of cancer cells in colon cancer suggests that a controlled up- regulation of a number of gene products is a charactenstic of budding colon cancer cells, and that these gene products serve functions crucial for the invasive phenotype of these cancer cells

A related aspect of the invention is the modulating the adhesion in vivo of cells presenting an integπn receptor recognized by the polypeptide of the invention Pharmaceutical compositions The invention provides pharmaceutical compositions compπsing a therapeutically effective dose of the polypeptide of the invention m a pharmaceutically acceptable excφient, for administration to a subject, such as a human patient The term "therapeutically effective amount" means the dose needed to effectively treat cellular infiltration and attendant cytokine network alterations associated with a vanety of inflammatory diseases and injunes For purposes of the invention, the terms "treat" or "treatment" include preventing, inhibiting, reducing the occurrence of, or ameliorating the physiological effects of the inflammatory condition treated

The term "pharmaceutically acceptable excφient" includes any solvents, dispersion media, antibacteπal and antifungal agents, microcapsules, hposomes, catiomc hpid earners, isotonic and absorption delaying agents and the like which are not incompatible with the active ingredients The formulation of pharmaceutical compositions is generally known in the art, reference can conveniently be made to Remington 's Pharmaceutical Sciences, 17th Edition (Mack Publishing Co , Easton, Pa ) Thus, the pharmaceutical compositions can compπse a suitable application medium, such as a gel, salve, lotion, colloid or powder, aqueous solutions such as physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as oli e oil or injectable organic esters Pharmaceutical compositions can be prepared as injectables, either as liquid solutions or suspensions The preparation can also be emulsified

Physiologically acceptable excipients include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients Suitable excipients are, for example, water, salme, dextrose, glycerol, ethanol, or the like and combinations thereof In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffenng agents that enhance the effectiveness of the active ingredient A polypeptide can also be formulated into the pharmaceutical composition as neutralized pharmaceutically acceptable salt forms

Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaπc, mandehc, and the like Salts formed with the free carboxyl groups can also be denved from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or feme hydroxides, and such organic bases as lsopropylamme, tnmethylamme, 2-ethylamιno ethanol, histidine, procame. and the like

The pharmaceutical forms suitable for infusion include steπle aqueous solutions or dispersions and stenle powders for the extemporaneous preparation of steπle injectable solutions or dispersion The form is fluid to the extent that easy synngabihty exists Typical excipients include a solvent or dispersion medium containing, for example, water buffered aqueous solutions (/ e , biocompatible buffers), ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants, or vegetable oils Stenhzation can be accomplished by any art-recognized technique, including but not limited to filtration or addition of antibactenal or antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid or thimerosal Further, isotonic agents such as sugars or sodium chlonde may be incorporated in the pharmaceutical compositions Prevention against microorganism contamination can be achieved through the addition of vaπous antibactenal and antifungal agents

Dosage The precise therapeutically effective amount of polypeptide of the invention used in the methods of this invention applied to humans can be determined by the ordmanly skilled artisan with consideration of individual differences in age, weight, extent of cellular infiltration by inflammatory cells and condition of the patient The pharmaceutical preparation of the invention should be administered to provide an effective concentration of 1 nM to 1 mM, preferably from 50 nM to 100 μM The M25 polypeptide has an IC,₀ = 25 μM

The concentration of a polypeptide of the invention required to obtain an effective dose in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered The total effective amount of a polypeptide of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short penod of time, or can be administered using a fractionated treatment protocol, in which the multiple doses are administered over a more prolonged penod of time

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage "Dosage unit form" refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated, each unit containing a predetermined quantity of active matenal calculated to produce the desired therapeutic effect in association with the required pharmaceutical excipient A pharmaceutical composition typically contains an amount of at least 0 1 weight % of active ingredient, i e , a polypeptide or antibody of this invention, per weight of total pharmaceutical composition A weight % is a ratio by weight of active ingredient to total composition Thus, for example, 0 1 weight % is 0 1 grams of polypeptide per 100 grams of total composition

Methods of administration The polypeptides of the invention can be administered in any way that is medically acceptable which may depend on the disease condition or injury being treated Possible administration routes include injections, by parenteral routes such as intravascular, intravenous, mtra-artenal, subcutaneous, intramuscular, mtratumor, mtrapeπtoneal, lntraventπcular, intraepidural or others, as well as oral, nasal, ophthalmic, rectal, topical, or by inhalation The compositions may also be directly applied to tissue surfaces during surgery Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants Administration can also be lntra-articularly, mtrarectally, intrapeπtoneally, intramuscularly, subcutaneously, or by aerosol inhalant in order to prevent particular inflammatory and immune reactions associated with such areas of the body Where treatment is systemic due, for example, to the presence in the subject of metastatic tumor cells, the composition can be administered orally or parenterally, such as mtrav enously, intramuscularly, subcutaneously, intraorbitally, mtracapsularly, intrapeπtoneally or intracisternally

The pharmaceutical compositions are conventionally administered intravenously, as by injection of a unit dose The term "unit dose" when used in reference to a pharmaceutical composition of the invention refers to physically discrete units suitable as unitary dosages for humans, each unit containing a predetermined quantity of active mateπal calculated to produce the desired therapeutic effect

The quantity to be administered depends on the subject to be treated, capacity of the subject to utilize the active ingredient, and degree of inhibition of receptor-hgand binding desired Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual However, suitable dosage ranges are of the order of one to several mg of active ingredient per individual per day and depend on the route of administration Suitable regimes for initial administration and booster shots are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration Alternatively, continuous intravenous infusions sufficient to maintain therapeutically effective concentrations in the blood are contemplated For a polypeptide of the invention, therapeutically effective blood concentrations are in the range of about 1 nM to 1 mM, preferably from 50 nM to 100 μM Whenever, the polypeptides of the invention are used for promoting attachment of cells, such compositions will typically have a higher concentration than those taken internally Use of polypeptides on a substrate for promoting cell attachment The polypeptide of the invention can be used in a composition for promoting the attachment (adhesion) of cells to a substrate Based on the ability of a polypeptide of the invention to bind with urokinase receptor on cells, the polypeptide provides a means for binding to the receptor to promote cell attachment activity when the polypeptide is immobilized onto a substrate In one aspect, the invention provides methods for attaching cells to a substrate in which the method compnses contacting cells expressing urokinase receptor with the substrate comprising the polypeptides of the invention affixed to a solid matnx and maintaining the contact for a predetermined time sufficient for the urokinase receptor to bind the polypeptides

The substrate can be any sohd-matnx having a surface on which cell adhesion promoting activity is desired and includes containers for cell culture, medical devices, prosthetic devices, synthetic resm fibers, blood vessels or vascular grafts, percutaneous devices, artificial organs, and the like The surface can additionally be compnsed of glass, a synthetic resin, nitrocellulose, polyester, agarose, collagen or a long chain polysacchaπde A composition containing a polypeptide of the invention is used to treat a substrate and thereby to immobilize the polypeptide contained m the composition onto the substrate Immobilization of polypeptides onto substrate can be accomplished by a vaπety of means and depends on the substrate and the mechanism of immobilization desired Methods for polypeptide immobilization or coupling to the substrate are well known in the art and typically involve covalent linkages between a thiol or amino group on the polypeptide to a reactive group present on the substrate For examples of polypeptide immobilization methods, see, United States patents 4,493,795, 4,578,079 and 4,671 ,950 In one aspect, the invention provides prosthetic and medical devices that make use of a substrate to attach cells to the surface in vivo or to promote growth of cells on a particular surface before to grafting For example, endothelial cell growth can be induced on prosthetic blood vessels or vascular grafts, such as those woven or knitted from polyester fibers For example, the polypeptides of the invention can be administered to the site of a wound, as a coating on a prosthetic device or incorporated into a matnx, thus mediating the binding of mtegnn-expressmg cells and increasing the rate of wound healing Methods of incorporating a polypeptide of the invention into an artificial extracellular matnx are descnbed in WO 90/06767

In such an aspect, the polypeptides can be usefully coupled to other biological molecules, such as collagen, glycosaminoglycans, etc The coupling can be facilitated by chemical cross-linking, e g , by disulfide bndges Surfaces of prosthetic devices can also be coated with the instant polypeptides, particularly when the devices are intended for use temporanly in the body, e g , for insertion into blood vessels or into the peπtoneal cavity Additional residues may also be added at either terminus of an polypeptide of this invention for the purpose of providing a "linker" by which the polypeptides of this invention can be conveniently affixed to a label or solid matnx, or earner Ammo acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues, but do not form epitopes cross-reactive with a M25 polypeptide of this invention Typical amino acid residues used for linking are tyrosine, cysteme, lysine, glutamic and aspartic acid, or the like In addition, a polypeptide of the invention can differ, unless otherwise specified, from the natural sequence of the coπespondmg protease by the sequence being modified by termmal-NFL acylation, e g , acetylation, or thioglycohc acid amidation, by termmal-carboxylamidation, e g , with ammonia, mefhylamine, and the like terminal modifications

Antibodies In one aspect, the normal cellular adhesion functions of a cell beaπng a cell surface urokinase receptor can be inhibited or modulated by administration of an effective amount of a pharmaceutically acceptable composition compnsing a polyclonal or monoclonal antibody that immunoreacts with a polypeptide of the invention Thus, an antibody directed against the polypeptide of the invention is also an aspect of this invention Polyclonal antibodies are produced by immunizing a mammal with a peptide immunogen Suitable mammals include primates, such as monkeys, smaller laboratory animals, such as rabbits and mice, as well as larger animals, such as horse, sheep, and cows Such antibodies can also be produced in transgenic animals However, a desirable host for raising polyclonal antibodies to a composition of this invention includes humans The polyclonal antibodies raised are isolated and punfied from the plasma or serum of the immunized mammal by conventional techniques Conventional harvesting techniques can include plasmapheresis, among others Such polyclonal antibodies can themselves be employed as pharmaceutical compositions of this invention Alternatively, other forms of antibodies can be developed using conventional techniques, including monoclonal antibodies, chimeπc antibodies, humanized antibodies and fully human antibodies (see e g , United States patent 4,376,1 10, Ausubel et al , Current Protocols in Molecular Biology (Greene Publishing Assoc and Wiley Interscience, N Y , 1992), Harlow & Lane, Antibodies a Laboratory Manual, (Cold Spnng Harbor Laboratory, 1988), Queen et al , 86 Proc Natl Acad Sci USA 10029-10032 (1989), Hodgson et al , 9 Bio/Technology 421 (1991), PCT International patent application WO 92/04381 and PCT

International patent application WO 93/20210 Other antibodies can be developed by screening hybπdomas or combinatoπal hbraπes, or antibody phage displays (Huse et al , 246 Science 1275-1281 (1988) using the polyclonal or monoclonal antibodies produced according to this invention and the amino acid sequences of the pnmary or optional immunogens "Antibodies" are lmmunoglobulm molecules and immunologically active fragments of immunoglobulm molecules Such portions known in the art as Fab, Fab', F(ab'), and F_v are included Antibodies can be polyclonal or monoclonal (mAb) Antibodies can bind a wide range of ligands, including polypeptides An "antibody combining site" is that structural portion of an antibody molecule that specifically binds (immunoreacts with) the polypeptide of the invention The term "immunoreact" refers to binding between an antigemc determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof An "antigemc determinant" is the actual structural portion of the antigen that is immunologically bound by an antibody combining site The term is also used interchangeably with "epitope" An "antigenically related vaπant" is a polypeptide that is capable of inducing antibody molecules that immunoreact with a polypeptide of this invention and immunoreact with M25

A therapeutically effective amount of an antibody of this invention is typically an amount of antibody such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0 1 μg/ml to about 100 μg/ml, and preferably from about 1 μg/ml to about 5 μg/ml

Screening The polypeptides of the invention can also be used for identifying and isolating cell types expressing certain cell surface receptors Cells expressing the urokinase receptor, for example, can be identified and isolated from a tissue by coating a surface or column with a peptide having specificity for the urokinase receptor, allowing the cells to bind to the surface or column, then eluting the cells from the surface or column, for example, using low pH buffer or EDTA-containing buffer Methods and cell lines for identification of regulators ofintegrin activation A selected cell line can be established which contains a functional urokinase receptor By "functional urokinase receptor" is meant a urokinase receptor capable of interacting with intracellular components and transmitting detectable information This functional urokinase receptor can be introduced into the selected cell line by transfection or may be endogenous to the selected cell line Cells of the cell line are then transfected with a chimeπc polypeptide having the cytoplasmic domain of an integπn subumt fused to a polypeptide containing extracellular and transmembrane domains that are not functional integnn domains, so that the chimera can inhibit signaling activities of the functional integπn by interaction with integnn regulator molecules in the cytoplasm The functional urokinase receptor and the chimera can each be introduced to the cell by a vanety of means well known to those of skill in the art, including but not limited to, by endogenous expression, by a transgene in the cell, or by transient expression of an expression vector A selected cDNA expression library is then introduced and expressed in the cell line Any integnn regulators expressed by the library will bind integnn cytoplasmic domains Overexpression of the integnn regulators will overcome the inhibition by the chimera, and signaling will occur, thus indicating expression of a functional regulator Drug design In one aspect, the invention is useful for designing drugs to modify integπn function which are targeted to urokinase receptor or to a region of the mtegnns to which the urokinase receptor bind Drugs whose structure is predicted based upon the molecular structure that interacts with the identified binding site can be designed Similarly, drugs identified as regulators of integπn function by the method of the invention or a region of the integπn to which they bind which will be useful in modifying mtegnn function can also be designed

Screening method for inhibitors of urokinase receptor/integrin activity The invention provides a method for screening for a urokinase receptor/integnn activity inhibitor The methods for assaying the ability of a compounds to inhibit the urokinase receptor/integnn activ lty are provided in the EXAMPLES The methods of the invention can easily be adapted for processing large numbers of samples, as is required in high throughput screening (HTS) settings at pharmaceutical and biotechnology companies Assays could be performed in 96 or 384 well plates using a minimal number of steps A vanety of colonmetπc, chemiluminescent, or fluorescent detection methods could be used to provide the sensitivity required for industrial applications

Compounds to be tested for urokinase receptor/integnn inhibitory activity can be hbranes (such as combmatoπal hbranes) of "small molecules," such as the term is understood by those of skill in the biochemical art Compounds to be tested for urokinase receptor/mtegπn inhibitory activity can be peptides, preferably peptides of less than 50 amino acids in length or fragments thereof The method of the invention can readily be adapted to a vanety of conditions, using standard methods

Secondary detection agents can be joined to a label, where the label can directly or indirectly provide a detectable signal Vaπous labels include fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e g magnetic particles, and the like Specific binding molecules include pairs, such as biotin and streptavidm, digoxm and antidigoxin For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures The secondary reagent can be an antibody coupled to horse radish peroxidase (HRP), fluorescent markers, streptavidm, or biotin Also, a vanety of other reagents may be included in the screening assay These include reagents like salts, neutral proteins, e g albumin, detergents, etc which are used to facilitate optimal protem-protem binding and/or reduce non-specific or background interactions Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors or anti-microbial agents may be used The mixture of components is added m any order that provides for the requisite binding Incubations are performed at any suitable temperature, typically between 4°C and 40°C Incubation penods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening Typically between 0 1 and 1 hours (hr) is sufficient

The details of one or more embodiments of the invention have been set forth in the accompanying descπption above Although any methods and matenals similar or equivalent to those descnbed herein can be used in the practice or testing of the present invention, the preferred methods and matenals are now descnbed Other features, objects, and advantages of the invention will be apparent from the descπption and from the claims In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs All patents and publications cited in this specification are incorporated by reference

The following EXAMPLES are presented in order to more fully illustrate the prefeπed embodiments of the invention These EXAMPLES should in no way be construed as limiting the scope of the invention, as defined by the appended claims

EXAMPLE 1 uPAR BINDS TO CELLS TRANSFECTED WITH Mac-1 iNTEGRLN, IDENTIFICATION

OF A uPAR BINDING SEQUENCE IN Mac-1

This EXAMPLE investigated the possibility that the urokinase receptor was restπcted m its interaction with members of the (CDl 1/CD18) family, t e CDl l a (LFA-1 , SEQ ID NO 12), CD l lb (Mac-1 , SEQ ID NO 1 1), CDl l c (p l 50,95 , SEQ ID NO 13), and CDl Id To explore this possibility and define the domains within the Mac-1 integnn responsible for this interaction, human urokinase receptor has been co-transfected into CHO cells expressing wild-type Mac- 1 or chimenc versions of Mac- 1 and p i 50,95

FIG 1 A schematically demonstrates that different chimeπc α subunits compnsed of various fusions of CD l lb (Mac-1 ) and CD l lc (p i 50,95) that all retain the ligand bmding I-domain of Mac- 1 (Diamond et al , 120 J Cell Biol 103 1 - 1043 (1993)) CHO cells transfected with human Mac-1 (Mac- 1 CHO), pi 50,95 (pi 50,95 CHO), or chimenc versions of Mac-1 and pi 50,95 were generated as previously descnbed by Diamond et α/ , 120 J Cell Biol 103 1 - 1043 (1993) β₂ integπn CHO cells were subsequently transfected with full length human urokinase receptor A polyclonal mixture of transfected cells was selected as a mixture of transfectants with a-MBM containing 10% FCS and 50 μ g/ml hygromycm 2 days after transfection Successful co-transfection of urokinase receptor into Mac- 1 CHO cells and Mac- 1/p 150,95 chimeπc CHO cells was confirmed by Western blotting and uPA binding Stable expression of a functional urokinase receptor was confirmed by western blotting analysis using rabbit anti-human uPAR antiserum and the demonstration of high affinity binding of [^l25I]urokinase (1 nM), as previously described by Wei et al., 269 J. Biol. Chem. 32380-32388 (1994). Stable clones were screened for expression by binding assays or flow cytometry. β₂ integrin (Mac-1, pi 50,95 and CDl lb/CD 1 lc chimeras)-transfected CHO cells with and without the urokinase receptor co-transfection were maintained as previously described by Simon et al., 88 Blood 3185-3194 (1996) and Wei et al., 269 J. Biol. Chem. 32380-32388 (1994)).

FIG. 1 A schematically illustrates different chimeric a subunits comprised of various fusions of CDl lb (Mac-1) and CDl lc (pi 50,95) that all retain the ligand binding I-domain of the Mac-1 integrin . For simplicity, we have renamed the original designations formulated by Diamond et al., 120 J. Cell Biol. 103 1-1043 (1993) as M (X-e-M), MP2 (M-b-X), MP3 (M-a-X), MP4 (X-e-M-b-X), and MP5 (M-b-X-a-M).

The degradation of [¹²T]-fibrinogen by β₂ integrin-transfected CHO cells was examined by the method reported by Simon et al., 88 Blood 3185-3194 (1996)). To investigate the effect of urokinase receptor occupancy on fibrinogen binding and degradation, incubations with transfected CHO cells were performed in the presence of ATF (5 nM). CHO cells transfected with the Mac-1 integrin bound, internalized, and degraded fibrinogen at a rate of 6.0 ± 2.8 pg fibrinogen/ 10⁶ cells/hr. Fibrinogen degradation by Mac-1 -transfected CHO cells is inhibited by LPM19c (% inhibition =90±14%), a mAb that blocks fibrinogen binding to the Mac-1 integrin confirming the role of Mac-1 in this degradation pathway. Relative binding/degradation was calculated as the ratio of the rate of binding/degradation in the presence of AU to the rate of binding/degradation in the absence of ATF.

The binding of [¹²T]-fibrinogen to THP-1 monocytic cells was investigated as described by Simon et al., 88 Blood 3185-3194 (1996), with modification. THP-1 monocytic cells were obtained from (American Type Culture Collection, Rockville, MD). Human plasminogen and plasminogen-free fibrinogen were purchased from Enzyme Research Laboratories, South Bend, IN. Fibrinogen and urokinase were iodinated, as described by Simon et al., 88 Blood 3185-3194 (1996). Briefly, THP-1 cells in RPMI containing 10% FCS were added to 96-well microtiter plates (lOVwell) and stimulated overnight with PMA (17 μ g/ml). Adherent wells were washed with RPMI containing 0.5% BSA, and [¹²⁵I]-fibrinogen (1.0 μM) was added to each well. After 60 minutes at 4°C, cell-bound fibrinogen was determined by washing the adherent cells, which were then lysed and counted. Specific binding was calculated by subtracting nonspecific binding in the presence of a 20-fold excess of unlabeled fibnnogen from total binding or by incubating cells in the presence of the Mac-1 blocking mAb, LPM 19c

Fibπnogen binding and degradation by CHO cells transfected with Mac-1 is inhibited by LPM 19c, a mAb that blocks fibnnogen binding to the Mac-1 mtegnn , confirming the role of Mac-1 in this degradation pathway LPML9c, a monoclonal antibody (mAb) to the -subunit of the Mac-1 integrm that blocks fibnnogen binding to Mac-1 , was purchased from DAKO Corp (Carpintena, CA) CHO cells transfected with vaπous chimeπc integπns, internalized, and degraded fibnnogen in a Mac- 1 -dependent manner (TABLE 3) The addition of ATF to Mac-1 /uP AR CHO cells blocked fibnnogen turnover by 79%±2% Inhibition by ATF was preserved in the Mac-l/pl50,95 CHO cell chimera designated MP3 (% ιnhιbιtιon=71 ± 10%)

-S I-

TABLE S

Effect of uPAR occupancy on LPM19c-ιnhιbιtable fibnnogen turnover m

Mac-l/pl 50,95 chimenc CHO cells transfected with human uPAR

α subunit FGN Turnover - FGN Turnover + % inhibition

ATF ATF (+ATF v -ATF)

(%Mac-l/uPAR (%Mac-l/uPAR

CHO) CHO)

Mac-1 100 ± 12 21 ± 2* 79 MP-1 122 ± 13 82 ± 25 33 MP-2 95 + 10 101 ± 18 0 MP-3 133 ± 12 38 ± 10** 71 MP-4 1 10 ± 10 1 16 ± 17 MP-5 95 ± 18 105 ± 14 0

The effect of urokinase receptor occupancy by ATF on fibnnogen binding and degradation by transfected CHO cells was investigated as descnbed m Methods

Equivalent uPAR expression in uPAR-transfected Mac-l/pl50,95 chimeras was venfied by specific ^l2:> I-urokinase binding ATF (5 nM) was pre-incubated with the cells for 30 minutes pπor to the addition of [^12:Tj-fibnnogen fibnnogen turnover inhibitable by the anti-CDl lb I-domain mAb LPM 19c was calculated and expressed relative to

Mac-1/uPAR CHO cells in the absence of ATF Values represent mean ± SEM (n=3-5) *

P <0 01 , **E' < 0 01

This inhibitory pattern suggested a possible interaction site between the urokinase receptor and the Mac-1 integnn in a region between the I-domain and the highly conserved divalent cation repeats of CDl lb (see, FIG IB) Inspection of this sequence revealed a previously unrecognized homology to peptide 25 (STYHHLSLGYMYTLN, SEQ ID NO 8), a uPAR-bindmg peptide previously identified in a phage display library by Wei et al , 273 Science 155 1-1555 (1996) Peptide 25 has about 33%o homology with the M25 polypeptide

We have termed this corresponding Mac- 1 sequence (α^M424-440, PRYQHIGLVAMFRQNTG, SEQ ID NO 1) the M25 polypeptide Homologies to the conespondmg sequences in LFA-1 (PRYQHMGRVLLFQEPQG, SEQ ID NO 2, "L25") and pi 50,95 (PRYQHTGKAVIFTQVSR, SEQ ID NO 3, "P25") are also shown in FIG IB EXAMPLE 2 uPAR BINDS TO M25 POLYPEPTIDE

To determine if suPAR actually bound to M25, solid-phase binding assays were performed with immobilized M25 polypeptide

Peptide amides were synthesized and punfied by Quality Controlled Biochemicals, Inc (Hopkmton, MA) All peptides were dissolved in DMSO at 1040 mM Nunc Maxisorb 96-well microtiter plates were coated with M25 (10 μg/ml) in PBS overnight at 37°C, and then blocked with the PBS containing 1% BSA Biotmylated suPAR (100 nM), in the presence or absence of M25 (PRYQHIGLVAMFRQNTG, SEQ ID NO 1), ScM25 (HQIPGAYRGVNQRFTML, SEQ ID NO 5), L25 (PRYQHMGRVLLFQEPQG, SEQ ID NO 3), or P25 (PRYQHTGKAVIFTQVSR, SEQ ID NO 2) (1-50 μM), was then added to each well for 1 5 hr at 25^υC After washing, avidm peroxidase was added and biotmylated suPAR binding quantified as descπbed above Relative binding was calculated as the ratio of binding in the presence of peptide to binding in the absence of peptide

Biotmylated suPAR (100 nM) was added to microtiter wells coated with M25 (10 μg/ml) and blocked with BSA

Results showed that biotmylated suPAR (100 nM) bound to the M25 polypeptide This bmding was blocked by soluble M25 (IC₅₀ -2 pM) but not by the scrambled version of M25, the ScM25 polypeptide (see FIG 2A) The specificity of suPAR-M25 interaction was explored further using soluble peptides conespondmg to sequences from LFA-1 (L25) or pi 50,95 (P25) L25 and P25 had no inhibitory effect on suPAR binding to M25, indicating that suPAR interacts preferentially with this Mac-1 sequence

The capacity of uPAR-expressing cells to attach to immobilized M25 was also tested Adherent cells were assayed by colonmetπc method of Simon et al , 88 Blood 3185-3194 (1996), or by loading cells with BCECF AM (1 μM) as descπbed by the manufacturer Cells (lO⁵ per well) were placed in 96-well microtiter plates coated with vitronectin (10 μg/ml), fibnnogen (100 μg/ml), or fibronectin (20 pg/ml) For adhesion expenments to peptides, wells were coated with peptides (80 μg/ml) in PBS overnight at 37°C and then blocked with the PBS containing 1% BSA Adhesion was stimulated with PMA (17 ng/ml) or the β₂-stιmulatιng KIM 127 mAb (5 μg/ml) Plates were washed with 0 9%o NaCl (3-5 times) and adherent cells were fixed in methanol for 15 mm, stained with Giemsa, and adhesion was quantified by measunng absorbance at 540 nm Alternatively, adhesion was quantified by measunng the fluorescence of BCECF AM-loaded cells using a Cytofluor II fluorescence multi-well microplate reader (PerSeptive Biosystems, Framingham, MA)

The monocytic cell line THP-1 expresses the urokinase receptor in response to stimulation with cytokines and exhibits urokmase-dependent adhesion to the matnx protein vitronectin Cytokine-stimulated THP-1 cells bound to immobilized M25 in a urokmase-dependent manner but did not attach to either ScM25, L25 or P25 (see, FIG 2B) Attachment to M25 was stimulated by either 10 nM active urokinase or Ar Binding was blocked by pnor treatment of the cells with phosphomositol-specific phosphohpase C, which removes most cell surface urokinase receptor and by a polyclonal anti-uPAR antibody, 399R (Polyclonal anti-uPAR antibody (399R) was from Amencan Diagnostica The monoclonal anti-uPAR antibodies LU and R3 were as descnbed by (Ronne et al 288 FEBS Letter 233- 236 (1991 )) Binding was not inhibited by two monoclonal uPAR antibodies known to bind domains β of the urokinase receptor (FIG 2B) Preferential binding of uPAR-transfected 293 cells as compared with non-transfected 293 cells to immobilized M25 was also observed Together, these observations indicate that the urokinase receptor directly binds a linear sequence in the Mac-1 integπn and this binding is promoted by urokinase

EXAMPLE 3 M25 DISRUPTS COMPLEX FORMATION BETWEEN uPAR AND β, AND β, INTEGRINS

mAbs which block Mac-1 function or a uPAR binding phage display peptide 25 which disrupts integnn uPAR complexes greatly diminished (> 80%>) uPAR-dependent adhesion of PMA-stimulated monocytic cells to serum or vitronectin (Wei et al , 273 Science 155 1-1555 (1996), Simon et al , 88 Blood 3185-3194 (1996)) Having demonstrated that M25 binds the urokinase receptor, we tested the ability of M25 to compete with β, and β, integnn binding to the urokinase receptor, as we have previously reported for the phage display-generated peptide 25 (STYHHLSLGYMYTLN, SEQ ID NO 8, see FIG IB)

Addition of M25 (75 μM) to lysates of uPAR-transfected 293 cells completely disrupted co-precipitation of the urokinase receptor with β, mtegnns whereas a scrambled M25 (ScM25) had no effect In one assay, uPAR, β, integπns, and src-family kinases were co-immunoprecipitated by incubating lysates from uPAR-transfected 293 cell with anti-βl mAb (JB 1A) m the presence or absence of peptide M25 or scM25 (100 μM) The anti-βl mAbs included JB1A and AB1937 (Chemicon) The immunoprecipitates were blotted with polyclonal anti-uPAR antibody (399R), stπpped, and reblotted for Src family kinases and β, (AB 1937) A pan-Src family kinase antibody (Src2) was purchased from Santa Cruz Biotech BCECF AM (2',7'-bιs-(2-carboyethyl)-5-(and-6)-carboxyfluoresceιn, acetoxymethyl ester) was purchased from Molecular Probes (Eugene, OR)

Analogous to peptide 25, M25 blocked co-precipitation of src-family kinases with β, mtegnns In these cells association of src- family kinases with β, integπns is uPAR-dependent

The co-immunoprecipitation of β, mtegnns, the urokinase receptor, and Src family kinases was performed as reported by Wei et al , 144 J Cell Biol 1285-1294 (1999) UPAR transfected 293 cells were lysed on ice for 30 mm in RIPA buffer containing 50 mM Tns-HCl, pH 7 5. 150 mM NaCl, 1% deoxycholate, 0 1% SDS, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM phenylmethyl-sulfonyifluoπde, and leupeptin (10 μg/ml) After precleaπng with protein A-agarose, lysates were incubated with antibody to β, (JB1A) in the presence or absence of peptide M25 or scM25 (100 pM) The immunoprecipitates were blotted with antibody to the urokinase receptor (399R), stnpped, and reblotted for Src family kinases (Src2) and β, (AB 1937)

In identical expeπments we could not co-precipitate β₂ mtegnns and the urokinase receptor in THP-1 cells As uPAR/β, mtegnn co-precipitation in Tnton X-100 lysates requires the presence of caveohn, caveolm might not stabilize uPAR/β₂ mtegnn complexes in these cells Therefore, the effect of M25 on uPAR/β₂ mtegnn interaction was examined with puπfied proteins Binding of biotmylated suPAR to microtiter wells coated with punfied Mac-1 was inhibited by 25 μM M25 (% inhibition = 57±6) but not by ScM25 (FIG 3) Higher concentrations of M25 could not be tested because in these assays higher concentrations of M25 lead to high background binding of suPAR, presumably because M25 itself bound to plastic binds suPAR (FIG 2A)

EXAMPLE 4 EFFECTS OF M25 ON INTEGRIN BINDING TO EXTRACELLULAR MATRIX PROTEINS

The Mac-1 integπn and the urokinase receptor are both functionally and physically linked on cells Therefore, we tested the effects of M25, which disrupts the physical association between the urokinase receptor and the Mac- 1 mtegnn (FIG 3), on the cell attachment function of these receptors in monocytic THP-1 cells

First, the M25 polypeptide blocked Mac-1 -dependent adhesion of THP-1 cells to immobilized fibnnogen in a dose dependent manner (IC.,₀ -25 μM), but did not inhibit soluble fibrinogen binding to adherent THP- 1 cells In fact Mac- 1 -dependent binding of fibπnogen to THP-1 cells was enhanced several-fold m the presence of the M25 polypeptide M25 also inhibited Mac-1 -dependent adhesion of thioglycollate-ehcited muπne neutrophils to stimulated endothelial cells The control peptide ScM25, the LFA-1 peptide L25, and pi 50,95 peptide P25 had no anti-adhesive effects Second, results showed that PMA-stimulated THP-1 cell adhesion to vitronectin was completely blocked by M25 Although ScM25 and P25 had no inhibitory effect, weak inhibition of the conespondmg LFA- 1 peptide, which has the closest homology to M25, is discernible Interestingly, in these cells M25 had no effect on THP-1 cell attachment to fibronectin Third, THP-1 cells were stimulated with PMA and adhesion to fibπnogen-coated wells was assessed m the presence of 0-50 μM M25, ScM25, or 10 μg/ml anti-CD l lb mAb LPM19c Mac-1 -dependent fibnnogen binding was assessed by adding 1 μM [¹²³I]-fibnnogen to PMA-stimulated adherent THP- 1 cells in the presence or absence of indicated peptides (25 μM) Data was expressed as LPM19c-mhιbιtable fibπnogen binding Fourth, the effect of M25 or control peptides (50 μM) on the adhesion of thioglycolate-elicited munne neutrophils to TNF-α-stimulated endothelial cells was assessed The dependence of this adhesion on Mac- 1 was examined by incubating cells with the anti-CD l lb mAb Ml/70 (10 μg/ml) Data is expressed as % adhesion m the absence of peptides or Ml 70 M l 0, a rat anti-mouse CDl lb mAb, was obtained from ATCC Fluorescein isothiocyanate-conjugated goat anti-mouse IgG, F(ab')₂ antibody was obtained from Boehπnger Mannheim Corp (Indianapolis, IN)

For neutrophil adhesion expenments, low passage (1 to 3) human saphenous vein endothelial cells (HSVECs) were grown to confluence in 96-well microtiter wells and stimulated with tumor necrosis factor alpha (TNFα, 10 ng/ml) for 4 hr to upregulate ICAM-1 expression (Bevilacqua et al , 76 J Chn Invest 2003-201 1 (1985)) Thioglycolate-elicited muπne neutrophils. obtained as reported (Lu et al , 99 J Chn Invest 1340- 1350 (1997)), were loaded with BCECF AM for 30 min at 37°C, washed, and stimulated with fMLP (1 μM) prior to adding to HSVEC monolayers.

Results are shown in FIG. 4C.

EXAMPLE 5

EFFECTS OF M25 ON CELL MIGRATION

In addition to associating with the Mac-1 integrin in leukocytes, the urokinase receptor physically associates with β, integrins and regulates their function in epithelial cells and smooth muscle cells (Wei et al, 144 J. Cell Biol. 1285-1294 (1999), Wei et al, 273 Science 155 1-1555 (1996)). Concordant with its ability to disrupt uPAR β, integrin complexes, M25 also inhibited β, integnn function. Early passage human vascular smooth muscle cells (SMC), utilizing β, integrins (Wei et al., 273 Science 155 1-1555 (1996), 24), spread slowly on fibronectin in the presence of 75 pM M25 whereas ScM25 had no effect. Within 2 hr, however, spreading and adhesion of SMC on fibronectin were comparable. Based on these observations we tested the effects of M25 on haptotactic migration of SMC toward fibronectin and collagen (FIG. 5).

Haptotactic smooth muscle cell migration was examined as reported by Wei et al, 144 J. Cell Biol. 1285-1294 (1999)). In brief, passage 1-3 human saphenous vein smooth muscle cells (10³) were seeded into Transwell inserts (Becton Dickinson) pre-coated on the bottom with fibronectin (10 μg/ml), vitronectin (20 μg/ml) or collagen type 1 (20 μg/ml), and cultured overnight with or without peptide M25 (25- 100 mM) or ScM2S (100 mM). Cells on both sides of the filter were detached and counted. All assays were performed in triplicate and the data expressed as % inhibition by the peptides. In summary, M25 markedly inhibited SMC migration toward either fibronectin or collagen in a dose dependent manner, whereas ScM25 had no effect.

EXAMPLE 6 EFFECTS OF MODIFIED M25 POLYPEPTIDE

This EXAMPLE tests the effect of modified M25 polypeptide. The results of this EXAMPLE show that the peptide size can be longer than 15 amino acids.

A polypeptide was produced from the vector system (Invitrogen, San Diego, CA) was tested for activity in inhibiting neutrophil adhesion (see, FIG. 6). The assay system was similar to that in EXAMPLE 4 The ammo acid sequence of the peptide is

STYHHLSLGYMYYLNGGEQKLISEEDL ("25myc", SEQ ID NO 9) and has a carboxy- terminal myc sequence

EXAMPLE 7

EFFECTS OF A325 ON HUMAN BREAST CANCER CELLS

This EXAMPLE tests the effect of the A325 polypeptide PRHRHMGAVFLLSQEAG, SEQ ID NO 4) on the adhesiveness of human breast cancer cell line 231 Using the assay system descπbed above for the M25 polypeptide, we found that the A325 polypeptide (10-50 μM) inhibits the migration of 231 cells The effects are similar to those measured for the effects of M25 polypeptide on smooth muscle cells (descπbed above)

These results show that disruption of the integnn/urokinase receptor interactions can be a therapeutic strategy for blocking metastasis The attractive feature of this therapy is that the A325 polypeptide (or structurally and functionally similar agents) would not greatly affect normal, non-tumoπgemc cells

EXAMPLE 8 THE EFFECT OF M25-DERIVED "LOOP" PEPTIDES ON UPAR-DEPENDENT 293 CELL ADHESION TO VITRONECTIN

The purpose of EXAMPLE is to form the basis for lead compounds m the in vivo use of peptides that bind to urokinase receptor

On the basis of homology with G-protem-coupled receptors, Spnnger, 94 Proc Natl Acad Sci US A 65-72 ( 1997) has proposed that the N-termmal region (-450 ammo acids) of mtegnn α subunits folds into a β-propeller We examined the structure of the subunit of Mac- 1 m the Spnnger propeller model We determined that M25 has a W4 upper loop that extends through the third β strand of this repeat and into a lower loop

We tested the ability of shorter loop-like peptides to similarly bind the urokinase receptor and inhibit post-receptor mtegnn function Peptide 64 (LGAPRYQHI SEQ ID NO 14) coπesponds to the upper loop Peptide 65 (MFRQNTGMW, SEQ ID NO 15) coπesponds to the lower loop Both peptides are capable of modulating integnn function similar to full length M25 FIG 7 demonstrates the effect of "loop" peptides on uPAR 293 cell adhesion to vitronectin These smaller sized peptides are advantageously easier to cychze than longer peptides. The smaller sized is also advantageous for screening for new compounds that bind to urokmase receptor

EXAMPLE 9

SMALL MOLECULE INHIBITORS BASED ON THE 17 AMINO ACID INTEGRIN α-

SUBUNIT SEQUENCE

The goal of this EXAMPLE focuses on developing small molecule inhibitors that disrupt the uPAR-integπn interaction and, thereby, modulate mtegπn function.

We tested the ability of two linear peptides (upper loop 64: LGAPRYQHI (SEQ ID. NO 14); lower loop: MFRQNTGMW (SEQ ID. NO' 15) that conespond to the predicted loop regions of the β-propeller model These peptides (9mer) are significantly shorter than the parent peptide (17mer) and are capable of modulating mtegπn function, inhibiting uPAR 293 cell adhesion to vitronectin. Based on these observations, we constructed cyclic peptides (cyclic 6mer designated c#l 17, cyclic 7mer designated c#293) using predicted C_β distances. The cyclic 7mer c#293 (CQNTGMC; SEQ ID NO: 16), but not the cyclic 6 mer c#l 17 (CQNTGC, SEQ ID NO: 17), inhibited integnn-dependent adhesion with an IC₅₀ (-25 μM) equivalent to the linear parent 17mer. (I) Screening series of peptides based on non-cell based purified protein binding assay We coat wells with M25 and assess the effect of increasing concentrations of candidate peptides and deπvatives ( 1 , 10, 25, 50, and 100 μM) on myc-uPAR binding to M25 Peptide sequences are generated based on structural information deπved from the β-propeller model

(2) Screening candidate peptides using cellular adhesion assay. Lead peptides are subjected to a cell-based assay. Inhibitory peptides identified from the bmding assay can be tested in a 96-mιcrotιter well adhesion assay using uPAR 293 cells.

(3) Determining the effect of mtegrin-derived peptides on in vivo models of inflammation We have established models of vascular injury and inflammation [Rogers et al., 95(17) Proc. Natl. Acad. Sci. USA 10134-9 (1998), Simon et al , 2000 (105) J. Chn. Inv 293- 3000 (2000), Simon et al , 192(2) J Exp. Med. 193-204 (2000)) to test the ability of candidate integπn peptides to modulate mtegnn function in expenmental angioplasty and lung injury. The foregoing description has been presented only for the purposes of illustration and is not intended to limit the invention to the precise form disclosed, but by the claims appended hereto

Claims

CLAIMS WE CLAIM:

1. An isolated polypeptide between 6 amino acids to 50 amino acids in length, comprising a polypeptide (a) wherein the peptide comprises 6 amino acid residue having greater than 40% homology with PRYQHIGLVAMFRQNTG (SEQ ID NO:l), as determined by the BLAST computer program; and (b) wherein the polypeptide binds specifically to the urokinase receptor (uPAR).

2. The polynucleotide of claim 1, wherein the polypeptide is derived from a W4 loop of an α integrin polypeptide.

3. The polynucleotide of claim 1, wherein the polypeptide comprises a peptide having an amino acid sequence PRXXHXGXVXXXXQXXG; SEQ ID NO:6), wherein X is an amino acid residue or a deletion.

4. The polynucleotide of claim 1 , wherein the polypeptide comprises a peptide having an amino acid sequence selected from the group consisting of PRYQHIGLVAMFRQNTG (SEQ ID NO: l); PRHRHMGAVFLLSQEAG (SEQ ID NO:4); LGAPRYQHI (SEQ ID NO: 14); MFRQNTGMW (SEQ ID NO: 15); and CQNTGMC (SEQ ID NO: 16).

5. The polypeptide of claim 1, wherein the polypeptide is cyclized.

6. The polypeptide of claim 1, in a pharmacologically acceptable excipient

7. A method for inhibiting complex formation between urokinase receptor (uPAR) and an integrin, comprising contacting a cell expressing the urokinase receptor (uPAR) with a polypeptide between 6 amino acids to 50 amino acids in length, (a) wherein the peptide comprises 6 amino acid residue having greater than

40% homology with PRYQHIGLVAMFRQNTG (SEQ ID NO: l), as determined by the BLAST computer program; (b) wherein the polypeptide binds specifically to the urokinase receptor (uPAR), and

(c) wherein contacting the cell inhibits the formation of complex formation between the urokinase receptor and the integnn

The method of claim 7, wherein the cells are selected from vascular endothelial cells, fibroblasts, smooth muscle cells, keratinocytes, placental trophoblasts, hepatocytes, tumor cells, and leukocytes

The method of claim 8, wherein the leukocytes are selected from the group consisting of T-cells, NK cells, monocytes, and neutrophils

The method of claim 7, wherein the integπn is selected from the group consisting of a β, mtegnn, a β₂ integnn, and a β₃ mtegnn

A method for inhibiting cell migration m a subject, compπsmg the step of admmistenng to a subject a polypeptide

(a) wherein the peptide compπses 6 ammo acid residue having greater than 40% homology with PRYQHIGLVAMFRQNTG (SEQ ID NO 1 ), as determined by the BLAST computer program,

(b) wherein the polypeptide binds specifically to the urokinase receptor (uPAR), and

(c) wherein the administered polypeptide inhibits cell migration in the subject

The method of claim 11, wherein the cell migration to be inhibited causes a condition selected from the group consisting of atherosclerosis, atherosclerosis, restenosis following percutaneous transluminal coronary angioplasty (PCT A), blood vessel disorders, transplant rejection, and obesity

The method of claim 12, wherein the administration is selected from the group consisting of oral, topical, parenteral, and intravascular administration A method for treating an acute or chronic inflammatory disorder in a subject, comprising the step of administenng to a subject with an acute or chronic inflammatory disorder a therapeutically effective amount of a polypeptide

(a) wherein the peptide compπses 6 amino acid residue having greater than 40% homology with PRYQHIGLVAMFRQNTG (SEQ ID NO 1 ), as determined by the BLAST computer program,

(b) wherein the polypeptide binds specifically to the urokinase receptor (uPAR), and

(c) wherein the administered polypeptide inhibits cell migration in the subject

A method for treating metastasis, compnsmg the step of administenng to a subject with a metastatic tumor a therapeutically effective amount of a polypeptide

(a) wherein the peptide compnses 6 ammo acid residue having greater than 40% homology with PRYQHIGLVAMFRQNTG (SEQ ID NO 1 ), as determined by the BLAST computer program, (b) wherein the polypeptide binds specifically to the urokinase receptor

(uPAR), and (c) wherein the administered polypeptide inhibits cell migration in the subject

A method of preventing integnn-mediated cell attachment to an extracellular matnx protein, compnsmg the step of contacting the cell expressing the integnn with a soluble urokinase receptor, wherein the contacting inhibits the binding of the integπn to the extracellular matrix protein

The method of claim 16, wherein the extracellular matnx protein is selected from the group consisting of vitronectin, fibnnogen, and collagen A method for screening for a compound that inhibits complex formation between urokinase receptor (uPAR) and mtegnn, compnsmg the steps of

(a) binding a urokinase receptor polypeptide to a polypeptide having greater than 40% homology with PRYQHIGLVAMFRQNTG (SEQ ID NO 1), as determined by the BLAST computer program, in the presence of compound suspected of inhibiting the formation of complex formation between the urokinase receptor and an integnn,

(b) binding a urokinase receptor polypeptide to the polypeptide having greater than 40%> homology with SEQ ID NO 1 in the absence of compound suspected of inhibiting the formation of complex formation between the urokmase receptor and an mtegnn, and

(c) companng the binding of the urokinase receptor polypeptide to a polypeptide having greater than 40% homology with SEQ ID NO 1 in step (a) with the binding m step (b), wherein a reduced binding m step (a) as compared with the binding in step (b) identifies the compound as being an inhibitor of complex formation between the urokinase receptor and mtegnn

A method for screening for a compound that inhibits complex formation between urokinase receptor (uPAR) and mtegnn, compnsmg the steps of

(a) contacting a cell expressing the urokmase receptor (uPAR) with a compound suspected of inhibiting the formation of complex formation between the urokinase receptor and an integπn, and

(b) companng the mtegnn-dependent activity of the cell in step (a) with a cell that has not been contacted with the compound, where a reduced mtegnn-dependent activity of the cell in step (a) as compared to the mtegnn-dependent activity of the cell that has not been contacted identifies the compound as being an inhibitor of complex formation between the urokinase receptor and integnn

The method of claim 19, wherein the mtegnn-dependent activity of the cell is selected from the group consisting of binding to extracellular matnx proteins and cell migration