WO1999065944A1 - PEPTIDE INHIBITORS OF αVβ3 AND αVβ¿5? - Google Patents

Abstract

The present invention includes peptides that bind to and inhibit the function of the integrin receptor, αVβ3. It also includes peptides that bind the integrin receptor αVβ5. The inventive peptides are between nine to about thirty amino acids in length. The peptides include an amino acid sequence comprising the binding domain Thr-Cys-X1-X2-Arg-Ala-Asp-Cys-X3 and an amino acid sequence comprising the binding domain Arg-Cys-Gly-Gly-Asp-Ser-X4-Cys-Tyr. These non-RGD peptides display surprisingly potent antagnonist activity despite the lack of the consensus binding sequence Arg-Gly-Asp, and present opportunities for selective targeting to the αVβ3 receptor. Pharmaceutical compositions and methods of use are also disclosed. The therapeutic uses for the inventive peptides include treating diseases involving αVβ3 receptors such as cancer, osteoporosis, restenosis, and angiogenic-based diseases.

Description

PEPTIDE INHIBITORS OF _vβ₃ and α_vβ₅

BACKGROUND

Field Of The Invention

The present invention relates to compositions for and methods of inhibiting integrin α_vβ₃ and/or α_vβ₅. Thus, the present invention relates to methods of treating and preventing α_vβ₃- and α_vβ₅- mediated diseases, particularly angiogenic diseases, by using certain novel o _vβ₃- specific peptides and novel peptides that bind _vβ₃ and α_vβ₅ . These peptides are also useful for diagnosing such diseases.

Background Art

The integrin receptor referred to as _vβ₃ serves as a receptor for a number of extracellular matrix proteins such as vitronectin, thrombospondin, von Willebrand factor, fibrinogen, fibrin and fibrinectin, and as such, can mediate various disease states. For example, it has been implicated in diseases such as angiogenic diseases (those pathological conditions that are affected by the proliferation of new blood vessels, e.g., cancer, diabetic retinopathy, and rheumatoid arthritis), osteoporosis (reduction in bone mass), and restenosis (migration and proliferation of smooth muscle cells). Certain antibodies to α_vβ₃ and certain peptides containing an R-G-D (Arg-Gly- Asp) sequence have been shown to bind to _vβ₃ and inhibit angiogenesis, osteoclast activity, and smooth muscle cell (SMC) migration and proliferation.

The growth of new blood vessels, or angiogenesis, involves α_vβ₃ and this process plays a key role in development, wound repair, and inflammation. The process also appears to contribute to pathological conditions such as diabetic retinopathy, rheumatoid arthritis, and cancer. There has been much interest in developing therapeutic agents that inhibit angiogenesis in these contexts and thus, treat disease states that are dependent on angiogenesis. Additionally, α_vβ₃ binding peptides may be used to identify the presence of the receptor and thus diagnose α_vβ₃ disease. The role of α_vβ₃ in the angiogenic process has been demonstrated using antibody and peptide inhibitors of this receptor. For example, antibodies to _vβ₃ blocked angiogenesis induced by basic fibroblast growth factor (bFGF), tumor necrosis factor (TNF-α) and human melanoma fragments, but had no effect on preexisting vessels. Brooks et al, Science, Vol. 264, pages 569-571 (1994). The authors of this study suggested that α_vβ₃ may be a useful therapeutic target for diseases characterized by neovascularization or angiogenesis.

The use of the α_vβ₃ receptor specific antibody, LM609, was also the subject of Brooks et al., Cell, Vol. 79, pages 1157-1164 (1994), where the antibody was shown to inhibit angiogenesis in a chicken embryo tumor model. Intravenous administration of the antibody caused tumor growth arrest and regression. A cyclic peptide containing the sequence Arg-Gly-Asp or RGD (cyclo-RGDfv) was also shown in this Cell publication to inhibit the neovascularization process in the chicken tumor model. These results support the earlier findings of Nicosia and Bonanno, Amer. J. Path., Vol. 138, pages 829-833 (1991), who showed in 1991 that the synthetic peptide Gly-Arg-Gly- Asp-Ser (GRGDS) (SEQ ID NO: 10) inhibits angiogenesis in vitro.

In addition to angiogenesis, the α_vβ₃ integrin has been demonstrated to play a role in smooth muscle cell migration. This has been shown using antagonists of the α_vβ₃ receptor including the LM609 antibody, and peptides GpenGRGDSPCA (SEQ ID NO:l 1) and N-mercapto acetyl D-Tyr-Arg-Gly-Asp-sulfϊde (Choi et al, J. Vase. Surg., Vol. 19, pages 125-134 (1994); Matusno et al, Circ. 90, pages 2203-2206 (1994), in in vitro and in vivo models of smooth muscle cell migration and restenosis. The specific vβ₃ inhibitors reduced neointima lesion formation, and the peptides which recognize vβ₃ and α_[Ibβ₃ also inhibited neointima formation in these models. Both groups concluded that the _vβ₃ receptor plays an important role in smooth muscle cell migration.

The involvement of the α_vβ₃ receptor smooth muscle cell migration in bone resorption has been demonstrated using antibody and peptide inhibitors of this integrin receptor. The α_vβ₃ receptor is expressed on osteoclasts, cells which mediate bone resorption through the matπx proteins osteopontm and bone sialoprotem (Miyauchi et al, J. Bwl Chem , Vol. 266, pages 20369-20374; Sato et al , J. Cell Biol, Vol. I l l, pages 1713-1723 (1990); Ross et al , J. Bwl Chem., Vol. 268, pages 9901-9907 (1993). In these publications, the α_vβ₃ specific LM609 antibody and Arg-Gly-Asp contaimng peptides blocked osteoclast attachment to and resorption on bone particles. These results supported early data by Horton et al (Exp. Cell Res , Vol 195, pages 368-375 (1991)) showing disruption of osteoclast function by the 23C6 α_v antibody and Arg- Gly-Asp containing peptide (GRGDSP) (SEQ ID NO: 12) Similarly Law et al, m J Clin Invest , Vol. 95, pages 713-724 (1995) confirmed the role of α,β, m the adherence and migration of cells due to the presence of osteopontm in an RGD-dependent manner.

As descπbed above, peptides that bind to the α_vβ₃ receptor have been demonstrated to inhibit the processes of angiogenesis, smooth muscle cell migration and osteoclast activity. In addition to their binding to the _vβ₃ receptor, these peptides have one other common determinant: they contain an RGD sequence

The RGD sequence has been shown to be essential for binding to the α_vβ₃ and other mtegπn molecules In 1984, Pierschbacher and Ruoslahti (Proc. Natl Acad. Sci , Vol. 81, pages 5985-5988) showed that the ammo acid sequence arginme, glycme, and aspartic acid or RGD is cπtical for the role of cell attachment activity essential for the mechanism of many integrals. The binding of α_vβ₃ and other mtegnns to RGD sequences is reviewed in Ruoslahti and Pierschbacher, Cell, Vol 44, pages 517-518 (1986) and discussed in Smith et al, J. Bwl Chem., Vol. 265, pages 12267-12271 (1990); Ruoslahti and Pierschbacher, Science, Vol. 238, pages 491-497 (1987);

Humphries, J. Cell Sci., Vol. 97, pages 585-92 (1990); Ruoslahti, J. Clin. Invest., Vol. 87, pages 1-5 (1991). Residues adjacent to the RGD can also play a role in binding as shown by Pierschbacher and Ruoslahti, 1987 (J. Bio. Chem., Vol. 262, pages 17924- 17928), and the process of cyc zing the RGD peptides can help to improve biological activity, Pierschbacher and Ruoslahti 1987 ibid; Aumailley et al, FEBS, Vol. 291, pages 50-54 (1991); and Koivunen et al, Biotech., Vol. 13, pages 265-270 (1995). Thus, a relationship between activity and conformation has been demonstrated for RGD peptides.

The necessary contribution of the RGD sequence to the inhibitory activity of these peptides for integrins has been demonstrated. Deletion or mutations of the RGD has been shown to abolish activity in Obara et al, Cell, 53, pages 649-657 (1988); Beacham et al, J. Biol Chem., Vol. 267, pages 3409-3415 (1992); and Cherny et al, J. Biol Chem., Vol. 268, pages 9725-9729 (1993). In fact, variations of RGD peptides such as cyclo RAD and RGE peptides have been traditionally used as negative controls for RGD activity (e.g., Brooks, et al, 1994, ibid, and Nicosia and Bonanno, 1991 ibid.). Additionally, when random cyclic libraries of peptides are screened with various integrins, including α₅β,, α_vβ₃, and α_libβ₃, RGD specific peptides with a variety of ring sizes are identified (Koivunen et al, Biotechnology, Vol. 13, pages 265-270 (1995). Interestingly, when these authors examined the sequences of specific _vβ₃-binding peptides isolated from their library, four clones which displayed apparent RGD homologs were isolated. In three clones, the glycine residue was substituted by leucine and in 1 clone, serine was substituted for glycine. One of the RLD peptides was synthesized and measured in binding and cell adhesion assays. The peptide (ACPSRLDSPCG) (SEQ ID NO: 13) was only partially selective for the integrin α_vβ₃ and had an activity 100-1000 fold lower than an RGD peptide. Thus the activity of specific antagonists to inhibit the _vβ₃ receptor in angiogenesis, smooth muscle cell migration and osteoporosis has been demonstrated. Since RGD peptide antagonists can bind and inhibit the activity of a variety of integrin molecules, they may not represent the best diagnostic or therapeutic agents for diseases involving only the _vβ₃ receptor.

Some RGD sequences within a particular ligand, vitronectin for example, appear to bind to several different integrins such as _vβ₃, _vβ₅, α_vβ,, and α_IIbβ₃. In addition to peptides which bind α_vβ₃ and _vβ₅, generally, this invention also provides certain non- RGD containing peptides show that specific binding to _vβ₃ over other integrins such as vβ₅ and _Iϊbβ₃. It has now been discovered that certain small peptides of this invention bind to the vβ₃ and _vβ₅, integrin receptor and are useful, for example, in inhibiting angiogenesis, treating angiogenic-based diseases, osteoporosis and restenosis. This is particularly surprising in view of the fact that the peptides do not contain an RGD sequence.

SUMMARY OF THE INVENTION

The present invention includes peptides that bind to and inhibit the function of the integrin receptor, α_vβ₃. It also includes peptides that bind the integrin receptor α_vβ₅. The inventive peptides are between nine to about thirty amino acids in length. The peptides include an amino acid sequence comprising the binding domain Thr- Cys-X,-X₂-Arg-Ala-Asp-Cys-X₃ and an amino acid sequence comprising the binding domain Arg-Cys-Gly-Gly-Asp-Ser-X₄-Cys-Tyr. These non-RGD peptides display surprisingly potent antagonist activity despite the lack of the consensus binding sequence Arg-Gly-Asp, and present opportunities for selective targeting to the _vβ₃ receptor. Pharmaceutical compositions and methods of use are also disclosed. The therapeutic uses for the inventive peptides include treating diseases involving α_vβ₃ receptors such as cancer, osteoporosis, restenosis, and angiogenic-based diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the inhibition of binding of vitronection to (A) _vβ₃ and (B) _vβ₅ by peptides. Absorbance of bound isotinyloted vitronectin was measured at 560 nm and graphed against concentration of peptide added. Peptides added were ( — □ — ) TCECRADCYC (SEQ ID NO: 2); ( ^{• ■••}<>^•••• ) TCSPRADCA (SEQ ID NO: 3); ( -^•o— )

RCGGDSMCY (SEQ ID NO: 4); ( Δ ) RCGGDSDCY (SEQ ID NO: 5); ( - -

. . □ ) GPenGRGDSPCA (SEQ ID NO: 6); ( — -0-— ) no inhibitor; and ( ® ) TCSPRAECAA (SEQ ID NO: 7). DETAILED DESCRIPTION OF THE INVENTION Definitions

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. Generally, the nomenclature used herein and the laboratory procedures described below are those well known and commonly employed in the art. Standard techniques are used for peptide synthesis recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art. The nomenclature used herein and the laboratory procedures in analytical chemistry, organic synthetic chemistry, and pharmaceutical formulation described below are those well known and are used as commonly employed in the art, unless indicated otherwise. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical formulation and delivery, and treatment of patients, unless otherwise indicated.

The term "peptide" as used herein means two or more amino acids that are linked together via a peptide bond.

In the formulas representing selected specific peptide embodiments of the present invention, the amino- and carboxy-terminal groups, although often not specifically shown, will be understood to be in the form they would assume at physiological pH values, unless otherwise specified. Thus, the N-terminal H₂ ⁺ and C-terminal-O^" at physiological pH are understood to be present though not necessarily specified and shown, either in specific examples or in generic formulas. In the peptide notation used herein, the lefthand end of the molecule is the amino terminal end and the righthand end is the carboxy-terminal end, in accordance with standard usage and convention. Of course, the basic and acid addition salts including those which are formed at nonphysiological pH values are also included in the compounds of the invention

The term "amino acid" as used herein includes the standard twenty genetically- encoded amino acids and their corresponding stereoisomers in the "D" form (as compared to the natural "L" form), omega-ammo acids, other naturally-occurring ammo acids, unconventional ammo acids (e g , - disubstituted ammo acids, N-alkyl ammo acids, etc ) and chemically deπvatized ammo acids When an ammo acid is being specifically enumerated, such as "alanme" or "Ala" or "A" the term refers to both L- alanme and D-alanine unless explicitly stated otherwise Other unconventional ammo acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide For the peptides shown, each encoded amino acid residue, where appropπate, is represented by a one- letter designation or a three-letter designation, corresponding to the trivial name of the conventional ammo acid

In keeping with standard peptide nomenclature (descπbed in J Biol Chem , 243 3552-59 (1969) and adopted at 37 CFR §1 822(b)(2) and (p)(2)), abbreviations for the 20 genetically-encoded, naturally-occumng amino acid residues are shown m the following Table I

TABLE I

AMTNO ACTD SYMBOL

3 -Letter 1 -Letter alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys C glutamic acid Glu E glutamine Gin Q glycine Gly G histidine His H isoleucine He I leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan Tip W tyrosine Tyr Y valine Val V

Abbreviations for Certain modified and unusual amino acids as shown in Table II and are also included within the definition of "amino acid."

Table II

Abbreviation Modified and unusual amino acid

Aad 2-Aminoadipic acid bAad 3-Aminoadipic acid bAla beta-Alanine, beta-Aminopropionic acid

Abu 2-Aminobutyric acid

4Abu 4-Aminobutyric acid, piperidinic acid

Acp 6-Aminocaproic acid

Ahe 2-Aminoheptanoic acid

Aib 2-Aminoisobutyric acid bAib 3-Aminoisobutyric acid

Apm 2-Aminopimelic acid

Dbu 2,4-Diaminobutyric acid

Des Desmosine

Dpm 2,2'-Diaminopimelic acid

Dpr 2.3-Diaminopropionic acid

ElGly N-Ethylgilycine

EtAsn N-Ethylasparagine

Hyl Hydroxylysine

AHyl allo-Hydroxylysine

3Hyp 3 -Hydroxypro line

4Hyp 4-Hydroxyproline

Ide Isodesmosine

Alle allo-Isoleucine

MeGly N-Methylglycine, sarcosine

Melle N-Methyliso leucine

MeLys N-Methylavaline

Nva Norvaline Table II - Cont... Abbreviation Modified and unusual amino acid

Nle Norleucine

Orn Ornithine

Certain commonly encountered amino acids, which are not encoded by the genetic code and not shown in Tables I or II, include, for example, other omega-amino acids, such as 3-amino propionic acid, sarcosine (Sar), citrulline (Cit), homoarginine (Har), t-butylalanine (t-BuA), t-butylglycine (t-BuG), phenylglycine (Phg), cyclohexylalanine (Cha), cysteic acid (Cya); pipecolic acid (Pip), thiazolidine (Thz), 2- naphthyl alanine (2-Nal) and methionine sulfoxide (MSO). Examples of unconventional amino acids include: γ-carboxyglutamate, e-N,N,N-trimethyllysine, e- N-acetyllysine, 0-phosphoserine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5 -hydroxylysine (Hyl), ω-N-methylarginine, and other similar amino acids and imino acids.

It is to be understood that in each of the formulae representing peptides of this invention shown herein, the peptides are cyclical, e.g. having Cys-Cys disulfide bonds forming the cyclic portion. This is even though the formula may not show a connecting line between the Cys moieties. Thus, the formula [SEQ. ID NO. 14]

Arg-Cys-Asp-Gly-X,-Cys-X,₊₂ is the same as the formula

Arg-Cys-Asp-Gly-X,-Cys-X₁₊₂; the formula

Arg-Cys-X,-Gly-Asp-X_rf-X,₊₄-Cys-X,₊₆ [SEQ ID NO. 15]

is the same as the formula

Arg-Cys-X_rGly-Asp-X₁₊₃-X₁₊₄-Cys-X₁₊₆, the formula (AA_N)_m-X_rCys-X₃-X₄-Arg-X₆-Asp-Cys-X₉-(AA_c)_n [SEQ. ID NO. 1] is the same as the formula

(AA_N)_m-X₁-Cys-X₃-X₄-Arg-X₆-Asp-Cys-X₉-(AA_c)_n.

Non-interfering substituents can be added to free functional groups, including those at the carboxy- or amino-terminus, by amidation, acylation or other substitution reactions. Such substitutions can, for example, change the solubility of the compounds without affecting their activity.

The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker, e.g. , by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods) . Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes (e.g., 3H, 14C, 35S, 1251, 1311), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g. , horseradish per oxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g. , leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential stearic hindrance.

As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85% , 90% , 95% , and 99% . Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

The present invention also provides peptide mimetics for the disclosed peptides. A "peptide mimetic" is defined to include a chemical compound, or an organic molecule, or any other peptide mimetic, the structure of which is based on or derived from a binding region of a protein. For example, one can model predicted chemical structures to mimic the structure of a binding region, such as a binding loop of a peptide. Such modeling can be performed using standard methods. In particular, the crystal structure of peptides and a protein can be determined by X-ray crystallography according to methods well known in the art. Peptides can also be conjugated to longer sequences to facilitate crystalization, when necessary. Then the conformation information derived from the crystal structure can be used to search small molecule databases, which are available in the art, to identify peptide mimetics which would be expected to have the same binding function as the protein (Zhao et al., 1995. "A paradigm for drug discovery using a conformation from the crystal structure of the presentation scaffold." Nat. Struct. Biol 2:1131-1137). The peptide mimetics identified by this method can be further characterized as having the same binding function as the protein according to the binding assays described herein.

Alternatively, peptide mimetics can also be selected from combinatorial chemical libraries in much the same way that peptides are. (Ostresh, J.M. et al, Proc NatlAcadSci USA 1994 Nov 8;91(23):11138-42; Dorner, B. et al, Bioorg Med Chem 1996 May;4(5):709-15; Eichler, J. et al, Med Res Rev 1995 Nov;15(6):481-96; Blondelle, S.E. et al Biochem J 1996 Jan 1;313 ( Pt l): 141-7; Perez-Paya, E. et al, J Biol Chem 1996 Feb 23;271(8):4120-6).

Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed. Parker, S. , 1985), McGraw-Hill, San Francisco, incorporated herein by reference).

Statement of Utility

A broad aspect of this invention is a physiologically acceptable non-RGD peptide of nine to thirty amino acid moieties which binds to the _vβ₃ integrin receptor. The peptides of this invention inhibit the function of the _vβ₃ integrin receptor in various disease states. Thus, the peptides may be combined with a pharmaceutically acceptable excipient to form a pharmaceutical composition useful for treating disease states or conditions that involve the α_vβ₃ integrin receptor as part of the disease process. The compounds also form the basis of a diagnostic method or kit for detecting a disease that involves the α_vβ₃ integrin receptor. The compounds of the invention can be used to screen for other compounds, including peptide and non- peptide (small molecule) compounds, that can compete for binding to _vβ₃ integrin receptor and/or _vβ₅ integrin receptor. The peptides of the invention, further, can be used to design and make mimetics, both peptide mimetics and non-peptide mimetics, that can mimic the binding of the peptides to _vβ₃ integrin receptor and/or to α_vβ₅ integrin receptor. Detailed Description and Preferred Embodiments

Compounds of the Invention

A. One aspect of this invention is a physiologically-acceptable peptide of five to about thirty amino acid moieties, wherein the peptide includes a cyclic portion that contains five to fourteen amino acid moieties, includes no -Arg-Gly-Asp- sequence, includes an amino acid sequence Arg-X₆-Asp- where X₆ is any naturally occurring amino acid except Gly and is positioned in the cyclic portion to bind to the _vβ₃ integrin receptor, and wherein the amino acid moieties external of the cyclic portion do not interfere with binding to the _vβ₃ integrin receptor. Preferably the cyclic portion contains six to eight amino acid moieties .

Another aspect of the invention is a physiologically-acceptable peptide of five to about thirty amino acid moieties, wherein the peptide includes a cyclic portion that contains five to fourteen amino acid moieties, includes no -Arg-Gly-Asp sequence, includes the amino acid sequence of Arg-X₆-Asp wherein X₆ is Ala, Ser, Thr, Asn, Leu, Gin, or Asp. The Arg-X₆-Asp sequence is positioned in the cyclic portion to bind to the _vβ₃ integrin receptor and the amino acid moieties external of the cyclic portion do not interfere with binding to the α_vβ₃ integrin receptor. Preferably the cyclic portion consists essentially of six to eight amino acid moieties.

As stated above, a broad aspect of this invention is a physiologically- acceptable peptide of five to about thirty amino acid moieties, wherein the peptide includes a cyclic portion that contains five to fourteen amino acid moieties, includes no -Arg-Gly-Asp- sequence, and includes an amino acid sequence Arg-X₆-Asp- where X₆ is any genetically-encoded, naturally-occurring amino acid except Gly. The cyclic portion is positioned to bind to an _vβ₃ integrin receptor and the amino acid moieties external of the cyclic portion do not interfere with binding to the _vβ₃ integrin receptor. The peptides as described where X₆ is Ala, Ser, Thr, Asn, Leu, Gin, Asp, Phe, Val, Lys, His, Met, Glu, Arg, lie, or Tyr are preferred, particularly those where X₆ is Ala, Ser, Thr, Asn, Leu, Gin, or Asp. The peptides where the cyclic portion contains six, seven, or eight amino acid moieties (especially seven) are particularly useful and therefore are also preferred. While the cyclic portion of the peptides may be established by any cyclizing mechanism, preferred formation is by Cys-Cys disulfide bonds so that there is at least one amino acid moiety external of the cyclic portion adjacent the Cys moiety on the N-terminus and at least one amino acid moiety external of the cyclic portion adjacent the Cys moiety on the C-terminus.

Thus, a preferred peptide in this family is one that contains at least two Cys moieties to form the cyclic portion by Cys-Cys disulfide bonds and that has at least one amino acid moiety external of the cyclic portion adjacent the Cys moiety on the N-terminus and at least one amino acid moiety external of the cyclic portion adjacent the Cys moiety on the C-terminus. Preferably the cyclic portion consists essentially of naturally-occurring, genetically-encoded amino acids and has six, seven or eight amino acid moieties (especially seven). Thus, a preferred peptide is a peptide that contains only two Cys moieties to form the cyclic portion by Cys-Cys disulfide bonds, wherein the cyclic portion contains seven moieties and X₆ is Ala, Ser, Thr, Asn, Leu, Gin, or Asp, and that has at least one amino acid moiety external of the cyclic portion adjacent the Cys moiety on the N-terminus and at least one amino acid moiety external of the cyclic portion adjacent the Cys moiety on the C-terminus. A preferred subgroup may be visualized as having the formula

(AA_N)_m-X_rCys-X₃-X₄-Arg-X₆-Asp-Cys-X₉-(AA_c)_n (I)

[ SEQ. ID NO. 1] wherein m is 0-11 and n is 0-11 with n+m < 21; and

AA_N is any amino acid at the N-terminus and AA_C is any amino acid at the C- terminus. Examples of representative peptides of this formula (I) include, for example, the following sequences (where the single capital letter refers to the L- amino acid residue shown in Table I and the single lower case letter refers to the D- amino acid residue corresponding to the L-amino acid of Table I):

TCECRADCYC

TCSPRADCAA TCSPRADCAa tCSPRADCAA

A particularly valuable subgroup is defined by formula wherein m and n each is 0 and X, and X₉ each is a naturally-occurring, genetically encoded amino acid or a corresponding D-isomer, preferably the latter. For ease of preparation it is preferable to have only two Cys moieties in the peptide, or if there are more than two Cys moieties, the additional Cys moieties are protected so that they do not form additional cyclic portions. More specifically, the peptides of formula (I) wherein X₃ is Ser or Asn, X₄ is Pro, and m and n are 0 are even more preferred. The following peptides where X_{ is Thr or Tyr and X₉ is Ala, Ser, or Tyr are of significant interest (e.g. where X₆ is Ala, Ser, Thr, or Leu):

Thr-Cys-Ser-Pro-Arg-X₆-Asp-Cys-Ala, [SEQ. ID NO. 16]

Thr-Cys-Ser-Pro-Arg-X₆-Asp-Cys-Ser, [SEQ. ID NO. 17] Thr-Cys-Ser-Pro-Arg-X₅-Asp-Cys-Tyr, [SEQ. ID NO. 18], or Tyr-Cys-Ser-Pro-Arg-X₆-Asp-Cys-Ser, [SEQ. ID NO. 19], more specifically

Thr-Cys-Ser-Pro-Arg-Ala-Asp-Cys-Ala, Thr-Cys-Ser-Pro-Arg-Ala-Asp-Cys-Ser, Thr-Cy s-Ser-Pro-Arg-Ala- Asp-Cy s-Tyr , or Tyr-Cys-Ser-Pro-Arg-Ala-Asp-Cys-Ser. Thr-Cys-Ser-Pro-Arg-Ser-Asp-Cys-Ser, Thr-Cys-Ser-Pro-Arg-Ser-Asp-Cys-Ser, Thr-Cys-Ser-Pro-Arg-Ser-Asp-Cys-Tyr, or Tyr-Cys-Ser-Pro-Arg-Ser-Asp-Cys-Ser, more specifically

Thr-Cys-Ser-Pro-Arg-Thr-Asp-Cys-Thr, Thr-Cys-Ser-Pro-Arg-Thr-Asp-Cys-Ser, Thr-Cys-Ser-Pro-Arg-Thr-Asp-Cys-Tyr, or Tyr-Cys-Ser-Pro-Arg-Thr-Asp-Cys-Ser, more specifically

Thr-Cys-Ser-Pro-Arg-Leu-Asp-Cys-Leu, Thr-Cys-Ser-Pro-Arg-Leu-Asp-Cys-Ser, Thr-Cys-Ser-Pro-Arg-Leu-Asp-Cys-Tyr, or Tyr-Cys-Ser-Pro-Arg-Leu-Asp-Cys-Ser, more specifically

Another specific subgroup includes those of formula (I) wherein m and n are both 0, X₃ is Glu, X₄ is Pro, X₆ is Ala, Ser, Thr, or Leu and X, is Thr and X₉ is Tyr, Ala, or Ser, e.g.

Thr-Cys-Glu-Pro-Arg-X₆-Asp-Cys-Ser, [SEQ. ID NO. 20] Thr-Cys-Glu-Pro-Arg-X₆-Asp-Cys-Tyr, [SEQ. ID NO. 21], or Thr-Cys-Glu-Pro-Arg-X₆-Asp-Cys-Ala, [SEQ. ID NO. 22]

particularly

Thr-Cys-Glu-Pro-Arg-Ala-Asp-Cys-Ser, Thr-Cy s-Glu-Pro-Arg- Ala- Asp-Cy s-Tyr , or Thr-Cys-Glu-Pro-Arg-Ala-Asp-Cys-Ala.

Thr-Cys-Glu-Pro-Arg-Thr-Asp-Cys-Ser, Thr-Cys-Glu-Pro- Arg-Thr-Asp-Cy s-Tyr , or Thr-Cys-Glu-Pro-Arg-Thr-Asp-Cys-Ala.

Thr-Cys-Glu-Pro-Arg-Leu-Asp-Cys-Ser, Thr-Cys-Glu-Pro-Arg-Leu- Asp-Cy s-Tyr, or

Thr-Cys-Glu-Pro-Arg-Leu-Asp-Cys-Ala.

Thr-Cys-Glu-Pro-Arg-Ser-Asp-Cys-Ser, Thr-Cys-Glu-Pro-Arg-Ser-Asp-Cys-Tyr, or Thr-Cys-Glu-Pro-Arg-Ser-Asp-Cys-Ala.

Still another more specific embodiment is the peptide wherein X, is Thr or Tyr, X₃ is Glu or Ser, X₄ is Cys (preferably protected to prohibit disulfide bond formation), and X₉ is Ala, Tyr, or Ser, e.g.

Thr-Cys-Ser-Cys*-Arg-X₆-Asp-Cys-Ala-Cys*, [SEQ. ID NO. 23] Thr-Cys-Ser-Cys*-Arg-X₆-Asp-Cys-Ser-Cys*, [SEQ. ID NO. 24] Tyr-Cys-Ser-Cys*-Arg-X₆-Asp-Cys-Ala-Cys*, [SEQ. ID NO. 25] Thr-Cys-Ser-Cys*-Arg-X₆-Asp-Cys-Tyr-Cys*, [SEQ. ID NO. 26] Tyr-Cys-Ser-Cys*-Arg-X₆-Asp-Cys-Tyr-Cys*, [SEQ. ID NO. 27]

Tyr-Cys-Ser-Cys*-Arg-X₆-Asp-Cys-Ser-Cys*, [SEQ. ID NO. 28] Thr-Cys-Glu-Cys*-Arg-X₆-Asp-Cys-Tyr-Cys*. [SEQ. ID NO. 29]

(wherein Cys* designates a Cys moiety that may be protected or unprotected and X₆ is Ala, Ser, Thr, or Leu), e.g.

Thr-Cys-Ser-Cy s *- Arg-Ala-Asp-Cy s-Ala-Cy s * ,

Thr-Cys-Ser-Cys*-Arg-Ala-Asρ-Cys-Ser-Cys*,

Tyr-Cys-Ser-Cys *- Arg-Ala-Asp-Cy s-Ala-Cy s * , Thr-Cys-Ser-Cys*-Arg-Ala-Asp-Cys-Tyr-Cys* , Tyr-Cys-Ser-Cys*-Arg-Ala-Asp-Cys-Tyr-Cys*, Tyr-Cys-Ser-Cys*-Arg-Ala-Asp-Cys-Ser-Cys*, Thr-Cys-Glu-Cys*-Arg-Ala-Asp-Cys-Tyr-Cys*

For any peptide, amino acid, or compound comprising an amino acid, of this invention, chemical derivatives of one or more A A members may be achieved by reaction with a functional side group. Such derivatized molecules include for example those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amino terminal acylation (e.g., acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. , with ammonia or methylamine), and the like terminal modifications.

Terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion and therefore to prolong the half-life of the peptides in solutions, particularly in biological fluids where proteases may be present. Polypeptide cyclization is also a useful modification and is preferred also because of the stable structures formed by cyclization and in view of the biological activities observed for cyclic peptides.

Another aspect of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a peptide of the invention.

Another aspect of the invention is a method of treating a disease that involves the α_vβ₃ integrin receptor, which method comprises administering a therapeutically effective amount of a peptide of the invention sufficient to bind to an α_vβ₃ receptor to treat or prevent such disease. Diseases for which this method can be used include angiogenic-based diseases, such as cancer, a retinal neovascular disease, diabetic retinopathy, an inflammatory disease, rheumatoid arthritis, psoriasis, and macular degeneration; diseases related to smooth muscle cell migration, such as restenosis, and cardiovascular disorders; and osteoporosis.

B. A broad aspect of this invention is a physiologically acceptable non-RGD peptide of five to thirty amino acid moieties, which peptide includes the partial sequence -Arg-Cys-Asp-Gly-X,- where X, is any amino acid moiety and the peptide includes a five-amino-acid cyclic portion. The cyclic portion is positioned to bind to the α_vβ₃ integrin receptor. The peptides of this invention inhibit the function of the α_vβ₃ integrin receptor in various disease states. Thus, the peptides may be combined with a pharmaceutically acceptable excipient to form a pharmaceutical composition useful for treating disease states or conditions that involve the α_vβ₃ integrin receptor as part of the disease process. The compounds also form the basis of a diagnostic method or kit for detecting a disease that involves the α_vβ₃ integrin receptor.

A preferred peptide in this family is one that contains at least two Cys moieties to form the cyclic portion by Cys-Cys disulfide bonds. A preferred subgroup may be visualized as having the formula (AA_N)_m-Arg-Cys-Asp-Gly-X_rCys-(AA_c)_n [SEQ. ID NO. 30] (II) wherein m is 0-12 and n is 0-13 with n+m < 24;

(AA_N)_m is any amino acid sequence at the N-terminus and (AA_c)_n is any amino acid sequence at the C-terminus.

A particularly valuable subgroup is defined by formula (II) wherein m and n each is 0, X, is any naturally-occurring, genetically encoded amino acid or a corresponding D-isomer, preferably the latter.

Another valuable subgroup may be visualized as having the formula

Arg- -Cys- Asp-Gly-X,-Cys-X, ₊₂ wherein X, is Pro or He; and

X_{] +2} is independently chosen from any naturally-occurring, genetically encoded amino acid.

The following peptides where X_{1 +2} is independently chosen from any naturally-occurring, genetically encoded amino acid is of significant interest (e.g. where X, is Pro or He):

Arg-Cys-Asp-Gly-X,-Cys-X_I+2, more specifically

Arg-Cys-Asp-Gly-Pro-Cys-X_{I +2} [SEQ. ID NO. 31] Arg-Cys-Asp-Gly-Ile-Cys-X,₊₂ [SEQ. ID NO. 32]

Another aspect of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a peptide of the invention.

Another aspect of the invention is a method of treating a disease that involves the _vβ₃ integrin receptor, which method comprises administering a therapeutically effective amount of a peptide of the invention sufficient to bind to an α_vβ₃ receptor to treat or prevent such disease. Diseases for which this method can be used include angiogenic-based diseases, such as cancer, a retinal neovascular disease, diabetic retinopathy, an inflammatory disease, rheumatoid arthritis, psoriasis, and macular degeneration; diseases related to smooth muscle cell migration, such as restenosis, and cardiovascular disorders; and osteoporosis.

C. Another aspect of this invention is a non-RGD nine amino acid cyclic peptide that binds to the α_vβ₃ integrin receptor. Another aspect of the invention is a non-RGD nine amino acid cyclic peptide that binds to the α,β₃ integrin receptor, wherein the cyclic portion contains 5, 6 or 7 amino acid moieties.

Another aspect of the invention is a non-RGD nine amino acid cyclic peptide wherein the cyclic portion includes the sequence, X,-Gly-Asp-, wherein X, is Gly, Ala, or Ser. A broad aspect of this invention is a nine amino acid peptide that preferably includes a cyclic portion having the sequence X,-Gly-Asp-, wherein X, is Gly, Ala, or Ser. This peptide is capable of binding to α_vβ₃ integrin receptor.

As stated above, an aspect of this invention is a nine amino acid peptide that includes a cyclic portion having the sequence X,-Gly-Asp-, wherein X, is Gly, Ala, or Ser. Aside from the sequence X_rGly-Asp in the 9 amino acid peptide wherein X, is Gly, Ala, or Ser, the other positions may be occupied by any amino acid. In general, however, it is preferred that the amino acids used in the cyclic peptide of this invention are chosen from the twenty, naturally-occurring, genetically-encoded amino acids. The cyclic portion can be formed from a disulfide linkage between two Cys residues of the peptide. The cyclic portion can be formed from a disulfide linkage between two Cys residues of the peptide.

Preferred compounds of the invention are presented by the formula Arg-Cys-X,-Gly-Asp-X₁₊₃-X₁₊₄-Cys-X₁₊₆ [(SEQ ID NO. 15] wherein X, is Gly, Ala, or Ser and X,^, X,₊₄, X₁₊₆ are any amino acid. Alternatively the formula can be rewritten to emphasize the cyclicity as

Arg-Cys-X_rGly-Asp-X₁₊₃-X,₊₄-Cys-X₁₊₆ wherein X, is Gly, Ala, or Ser and X₁₊₃, X₁₊₄, X₁₊₆ are any amino acid. Specifically preferred are peptides wherein X₁₊₃ is Ser; wherein X₁₊₄ is Met; wherein X,₊₆ is Thr; wherein X₁₊₆ is Tyr. A specifically preferred peptide is Arg-Cys-X,-Gly-Asp-Ser-Met- Cys-Thr (cyclized), particularly wherein X, is Gly, Ser, or Ala.

Preferred peptides also include:

Arg-Cys-Gly-Gly-Asp-Ser-X₁₊₄-Cys-X,_^6

Arg-Cys-Ala-Gly-Asp-Ser-X₁₊₄-Cys-X,₊₆

Arg-Cys-Ser-Gly-Asp-Ser-X₁₊₄-Cys-X,₊₆ Arg-Cys-Gly-Gly-Asp-Asn-X₁₊₄-Cys-X_]÷6

Arg-Cys-Ala-Gly-Asp-Asn-X₁₊₄-Cys-X₁₊₆

Arg-Cys-Ser-Gly-Asp-Asn-X₁₊₄-Cys-X₁₊₆

Arg-Cys-Gly-Gly-Asp-Tyr-X₁₊₄-Cys-X₁₊₆

Arg-Cys-Ala-Gly-Asp-Tyr-X_]+4-Cys-X_l46 Arg-Cys-Ser-Gly-Asp-Tyr-X₁₊₄-Cys-X,₊₆

Arg-Cys-Gly-Gly-Asp-Thr-X₁₊₄-Cys-X,.₆

Arg-Cys-Ala-Gly-Asp-Thr-X₁₊₄-Cys-X,^

Arg-Cys-Ser-Gly-Asp-Thr-X₁₊₄-Cys-X,₊₆

Arg-Cys-Gly-Gly-Asp-Asp-X₁₊₄-Cys-X,₊₆ Arg-Cys-Ala-Gly-Asp-Asp-X_]+4-Cys-X,₊₆

Arg-Cys-Ser-Gly-Asp-Asp-X_]+4-Cys-X₁₊₆

wherein X₁₊₄ and X₁₊₆ are any amino acid. Especially preferred are sequences wherein X_]+4 is any genetically encoded amino acid except Gly or He, and X₁₊₆ is any genetically encoded amino acid except Cys. A particularly preferred peptide includes the sequence Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr. Another aspect of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a peptide of the invention. In particular is a pharmaceutical composition comprising one or more of the following preferred peptides:

Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr Arg-Cys-Ser-Gly-Asp-Ser-Met-Cys-Tyr

Also provided in this invention is a diagnostic agent comprising a peptide of the invention. Preferred peptides for a diagnostic agent include:

Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr Arg-Cys-Ser-Gly-Asp-Ser-Met-Cys-Tyr

Another aspect of the invention is a method of treating a disease that involves the α_vβ₃ integrin receptor, which method comprises administering a therapeutically effective amount of a peptide of the invention sufficient to bind to an α_vβ₃ receptor to treat or prevent such disease. The peptides of this invention inhibit the function of the α_vβ₃ integrin receptor in various disease states. Thus, the peptides may be combined with a pharmaceutically acceptable excipient to form a pharmaceutical composition useful for treating disease states or conditions that involves the α_vβ₃ receptor. A preferred peptide for use in a method of treating a disease is Arg-Cys-Gly-Gly-Asp-Ser- Met-Cys-Tyr, cyclized. Diseases for which this method can be used include angiogenic-based diseases, such as cancer, retinal neovascular disease, e.g., diabetic retinopathy; an inflammatory disease, rheumatoid arthritis; psoriasis; and macular degeneration; diseases related to smooth muscle cell migration, such as restenosis; and cardiovascular disorders; and osteoporosis. D. The present invention provides an amino acid sequence comprising the binding domain Thr-Cys-X,-X₂-Arg-Ala-Asp-Cys-X₃ (SEQ ID NO: 8) which binds integrin vβ₃. By "binds" includes that the binding domain may additionally bind other targets, depending upon the amino acid selected for X„ X₂ and X₃. For example, when X, is Glu, X₂ is Cys, and X₃ is Tyr, the binding domain of the amino acid sequence binds both integrin α_vβ₃ and integrin α_vβ₅. However, this domain can also specifically bind integrin α_vβ₃, if such specificity is desired, depending upon the amino acid selected for X_{l 5} X₂ and X₃. For example, when X, is Ser, X₂ is Pro, and X₃ is Ala, the binding domain of the amino acid sequence specifically binds integrin _vβ₃. By "specifically binds" is meant that a binding domain selectively binds the target. That is, the peptide could be used to selectively remove the target (or the target used to selectively remove the peptide) from a sample containing the target (or peptide), or the peptide could be used to inhibit the target's biological activity. Such binding can readily be determined by radioimmunoassay (RJA), bioassay, or enzyme-linked immunosorbant (ELISA) technology. Thus, one can select the desired specificity of a peptide.

The present invention provides an amino acid sequence comprising the binding domain Thr-Cys-X,-X,-Arg-Ala-Asp-Cys-X₃ (SEQ ID NO: 8) which binds integrin α_vβ₃, wherein the amino acid sequence comprises nine to about thirty amino acid residues. The length of the amino acid sequence is only limited by the addition of additional amino acids that interfere in the binding of the binding domain to its target. For example, the amino acid sequence can have from 5 to about 50 amino acids, from 5 to about 75 amino acids, from 5 to about 100 amino acids, from 5 to about 200 amino acids, from 5 to about 300 amino acids, from 5 to about 500 amino acids. Typically, the binding domain contains a cyclic portion formed by a disulfide bond between two Cys residues in the binding domain. The invention specifically provides an amino acid sequence comprising the binding domain Thr-Cys-X_rX₂-Arg-Ala-Asp- Cys-X₃ (SEQ ID NO: 8) wherein X_! is Glu. The invention also specifically provides an amino acid sequence comprising the binding domain

Cys-X₃ (SEQ ID NO: 8) wherein X₂ is Pro. The invention further specifically provides an amino acid sequence comprising the binding domain Thr-Cys-X,-X₂-Arg- Ala-Asp-Cys-X₃ (SEQ ID NO: 8) wherein X₃ is Tyr.

Additionally, the invention specifically provides an amino acid sequence comprising the binding domain Thr-Cys-X _rX₂-Arg- Ala- Asp-Cy s-X₃ (SEQ ID NO: 8) wherein X, is Ser. The invention also specifically provides an amino acid sequence comprising the binding domain Thr-Cys-X,-X₂-Arg-Ala-Asp-Cys-X₃ (SEQ ID NO: 8) wherein X₂ is Cys. The invention further specifically provides an amino acid sequence comprising the binding domain Thr-Cys-X₁-X₂-Arg-Ala-Asp-Cys-X₃ (SEQ ID NO: 8) wherein X₃ is Ala.

Thus some embodiments of the present invention are the following peptides: Thr-Cys-Glu-X₂-Arg-Ala-Asp-Cys-X₃ Thr-Cys-Ser-X₂-Arg-Ala-Asp-Cys-X₃

Thr-Cys-X,-Cys-Arg-Ala-Asp-Cys-X₃ Thr-Cys-X_rPro-Arg-Ala-Asp-Cys-X₃ Thr-Cys-X,-X₂-Arg-Ala-Asp-Cys-Tyr Thr-Cys-X,-X₂-Arg-Ala-Asp-Cys-Tyr-Cys Thr-Cys-X,-X₂-Arg-Ala-Asp-Cys-Ala

Thr-Cys-Glu-Cys-Arg-Ala-Asp-Cys-Tyr-Cys (TCECRADCYC) [SEQ ID NO:2]

Thr-Cys-Ser-Pro-Arg-Ala-Asp-Cys-Ala (TCSPRADCA) [SEQ ID NO:3]

The present invention further provides an amino acid sequence comprising the binding domain Arg-Cys-Gly-Gly-Asp-Ser-X₄-Cys-Tyr (SEQ ID NO: 9) which binds integrin _vβ₃. Specifically the invention provides such a sequence wherein X₄ is Met. When X₄ is Met, the amino acid sequence binds both integrin _vβ₃ and integrin α_vβ₅. Specifically the invention also provides such a sequence wherein X₄ is Asp. When X₄ is Asp, the amino acid sequence binds both integrin α_vβ₃ and integrin _vβ₅. The present invention further provides an amino acid sequence comprising the binding domain Arg-Cys-Gly-Gly-Asp-Ser-X₄-Cys-Tyr (SEQ ID NO: 9) which binds integrin α_vβ₃, wherein the amino acid sequence comprises nine to about thirty amino acid residues. The length of the amino acid sequence is only limited by the addition of additional amino acids that interfere in the binding of the binding domain to its target. The binding domain contains a cyclic portion formed by a disulfide bond between two Cys residues in the binding domain.

Specific example of such peptides include the following:

Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr (RCGGDSMCY) [SEQ ID

NO:4]

Arg-Cys-Gly-Gly-Asp-Ser-Asp-Cys-Tyr (RCGGDSDCY) [SEQ ID

NO:5]

The present invention additionally provides a peptide mimetic of the amino acid sequence Thr-Cys-X_!-X₂-Arg-Ala-Asp-Cys-X₃ (SEQ ID NO:8). The present invention further provides a peptide mimetic of the amino acid sequence Arg-Cys- Gly-Gly-Asp-Ser-X₄-Cys-Tyr (SEQ ID NO: 9). The invention further provides a peptide mimetic of any of the above listed specific peptides.

Methods of use

The present invention provides a method of inhibiting binding of a ligand to integrin ocvβ3 expressed on a cell comprising administering to the cell an amino acid sequence comprising Thr-Cys-X_rX₂-Arg-Ala-Asp-Cys-X₃ (SEQ ID NO:8).

Specifically the method can be performed with an amino acid sequence comprising Thr-Cys-Glu-Cys-Arg-Ala-Asp-Cys-Tyr-Cys (SEQ ID NO:2). Specifically the method can also be performed with an amino acid sequence comprising Thr-Cys-Ser- Pro-Arg-Ala-Asp-Cys-Ala (SEQ ID NO:3). The present invention provides a method of inhibiting binding of a ligand to integrin αvβ3 expressed on a cell comprising administering to the cell an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-X₄-Cy s-Tyr (SEQ ID NO: 9). Specifically the method can be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr (SEQ ID NO:4). Specifically the method can also be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp- Ser-Asp-Cys-Tyr (SEQ ID NO: 5).

The present invention provides a method of inhibiting binding of a ligand to integrin α_vβ₅ expressed on a cell comprising administering to the cell an amino acid sequence comprising Thr-Cys-Glu-Cys-Arg-Ala-Asp-Cys-Tyr-Cys (SEQ ID NO:2). The present invention also provides a method of inhibiting binding of a ligand to integrin _vβ₅ expressed on a cell comprising administering to the cell an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-X₄-Cy s-Tyr (SEQ ID NO 9). Specifically the method can be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr (SEQ ID NO:4). Specifically the method can also be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp- Ser-Asp-Cys-Tyr (SEQ ID NO:5).

The present invention additionally provides a method of inhibiting binding of a ligand to integrin αvβ3 expressed on a cell in a subject comprising administering to the subject the amino sequence comprising Thr-Cys-X,-X₂-Arg-Ala-Asp-Cys-X₃ (SEQ ID NO: 8). Specifically the method can be performed with an amino acid sequence comprising Thr-Cys-Glu-Cys-Arg-Ala-Asp-Cys-Tyr-Cys (SEQ ID NO:2). Specifically the method can also be performed with an amino acid sequence comprising Thr-Cys-Ser-Pro-Arg-Ala- Asp-Cy s- Ala (SEQ ID NO:3).

The present invention additionally provides a method of inhibiting binding of a ligand to integrin αvβ3 expressed on a cell in a subject comprising administering to the subject an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-X₄-Cys- Tyr (SEQ ID NO: 9). Specifically the method can be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr (SEQ ID NO:4). Specifically the method can also be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-Asp-Cys-Tyr (SEQ ID NO:5).

The present invention further provides a method of inhibiting binding of a ligand to integrin α_vβ₅ expressed on a cell in a subject comprising administering to the subject an amino acid sequence comprising Thr-Cys-Glu-Cys-Arg-Ala-Asp-Cys-Tyr- Cys (SEQ ID NO: 2). The present invention further provides a method of inhibiting binding of a ligand to integrin α_vβ₅ expressed on a cell in a subject comprising administering to the subject an amino acid sequence comprising Arg-Cys-Gly-Gly- Asp-Ser-X₄-Cy s-Tyr (SEQ ID NO: 9). Specifically the method can be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr (SEQ ID NO:4). Specifically the method can also be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-Asp-Cys-Tyr (SEQ ID NO: 5).

Thus, the present invention provides both ex vivo and in vivo methods of use for the novel peptides. Details for administering the amino acid sequences, preferably in a composition including, for in vivo administration, a pharmaceutically acceptable carrier, are provided herein.

The present invention also provides a method of identifying a compound (e.g. , a peptide mimetic) which binds integrin _vβ₃ or α_vβ₅ comprising determining the crystal structure of the peptide of this invention, particularly those having the amino acid sequence: Thr-Cys-Glu-Cys-Arg-Ala-Asp-Cys-Tyr-Cys (SEQ ID NO: 2); identifying a compound having a structure which mimics the crystal structure of the peptide and detecting the ability of the compound to bind integrin _vβ₃ or _vβ₅.

The present invention also provides a method of identifying a compound (e.g., a peptide mimetic) which binds integrin _vβ₃ comprising determining the crystal structure of a peptide having the amino acid sequence: Thr-Cys-Ser-Pro-Arg-Ala- Asp-Cys-Ala (SEQ ID NO:3); identifying a compound having a structure which mimics the crystal structure of the peptide; and detecting the ability of the compound to bind integrin α_vβ₃.

The present invention also provides a method of identifying a compound (e.g., a peptide mimetic) which binds integrin α_vβ₃ or _vβ₅ comprising determining the crystal structure of a peptide having the amino acid sequence: Arg-Cys-Gly-Gly-Asp- Ser-Met-Cys-Tyr (SEQ ID NO: 4); identifying a compound having a structure which mimics the crystal structure of the peptide; and detecting the ability of the compound to bind integrin _vβ₃ or _vβ₅

The present invention also provides a method of identifying a compound (e.g., a peptide mimetic) which binds integrin _vβ₃ or _vβ₅ comprising determining the crystal structure of a peptide having the amino acid sequence: Arg-Cys-Gly-Gly-Asp- Ser-Asp-Cys-Tyr (SEQ ID NO: 5); identifying a compound having a structure which mimics the crystal structure of the peptide; and detecting the ability of the compound to bind integrin α_vβ₃ or α_vβ₅.

The compounds which bind integrins _vβ₅ and/or _vβ₃ can be a peptide mimetic, which can be a small molecule . The crystal structure of the peptides of this invention can be determined by X-ray crystallography procedures which are well known in the art. The crystal structure information can then be used to identify small molecules in a small molecule database according to standard methods (see, e.g., Zhao et al.). Such small molecules identified by these methods to have a structure which mimics the crystal structure of the peptide can be further identified as having the ability to bind vβ₃ and/or α_vβ₅ according to the binding assays described herein. Alternatively, a small molecule library can be produced according to standard methods and screened for the ability to bind _vβ₃ and/or α_vβ₅ according to the methods provided herein. The present method further provides a method of screening for a compound that binds integrin ocvβ3 comprising contacting αvβ3 with the compound and with a peptide of the invention, e.g. the amino acid sequence Thr-Cys-X,-X₂-Arg-Ala-Asp- Cys-X₃ (SEQ ID NO: 8), and determining whether the compound competitively inhibits the binding of the amino acid sequence to integrin vβ3. This method can be performed using, for example, Thr-Cys-Glu-Cys-Arg-Ala-Asp-Cys-Tyr-Cys (SEQ ID NO: 2 or Thr-Cys-Ser-Pro-Arg-Ala-Asp-Cys-Ala (SEQ ID NO:3).

The present invention also provides a method of screening for a compound that binds integrin ccvβ3 comprising contacting vβ3 with the compound and with the amino acid sequence Arg-Cys-Gly-Gly-Asp-Ser-X₄-Cys-Tyr (SEQ ID NO: 9) and determining whether the compound competitively inhibits the binding of the amino acid sequence to integrin vβ3. For example, the method can be performed with an amino acid sequence comprising Arg-Cys-Gly-Gly-Asp-Ser-Met-Cys-Tyr (SEQ ID NO:4) or Arg-Cys-Gly-Gly-Asp-Ser-Asp-Cys-Tyr (SEQ ID NO:5).

For these screening methods, in vitro competitive binding assays as described herein can be utilized.

Identification and Chemical Synthesis of the Invention Peptides

With the teachings of this specification in hand, one skilled in the art can understand how the peptide antagonists of α_vβ₃ are identified by panning phage- display peptide libraries with the receptor attached to a solid support, for example small diameter (1 μm) polystyrene latex beads. Phage selected by this method can then be tested for specific binding to α_vβ₃ or _vβ₅ via ELISA or other immunologically-based assays. Individual peptide sequences are then determined via sequencing of phage DNA. Further analysis of the minimal peptide sequence required for binding can be assessed via deletion and site-directed mutagenesis, followed by testing of the phage for binding to _vβ₃ or α_vβ₅ via ELISA. Since the identified peptide candidates are fused to the major phage coat protein, soluble peptides are then chemically synthesized and the activity of these free peptides tested in various in vitro and in vivo assays for the ability to act as antagonists of the α_vβ₃ receptor and α_vβ₅ receptor. A general discussion of epitope, or peptide, libraries by Jamie K. Scott is found at TIB517, July 1992, p 241-245. A specific process for synthesizing the oligonucleotides is set forth in U.S. Patent No. 5,264,563 issue November 23, 1993 to Huse. A related process is presented in an article by Koivunen, et. al. , J. Cell Bio, 124: 373-380 (1994). These are each incorporated herein by reference.

The general foregoing description may be modified to emphasize a preferred aspect of the invention, i.e. the presence of Cys groups to form Cys-Cys double bonds. Cysteine-doped phage-display peptide libraries are constructed using codon- based mutagenesis as per the Huse patent. These libraries consist of a mixture of approximately 10⁹ random peptides which are 20 amino acids in length and which have a greater than 1 in 20 chance of containing a cysteine residue at most positions. This provides for peptides which are biased toward being conformationally constrained by disulfide bonds and thus are more likely to bind with higher affinity to α_vβ₃ compared with linear peptides. Also, by using the codon-based mutagenesis technology, phage-display peptide libraries can be constructed which are biased toward peptides that lack the known tripeptide sequence RGD. This type of library increased the frequency of identifying non-RGD containing peptides. This is of some importance, since part of this invention is the discovery that non-RGD containing peptides can afford greater selectivity in binding to _vβ₃ over other integrin molecules which are known to interact well with proteins or peptides which contain an RGD tripeptide.

A peptide of this invention can be synthesized by several methods, including chemical synthesis and recombinant DNA techniques. Synthetic chemistry techniques, such as solid phase Merrifield synthesis are preferred for reasons of purity, freedom from undesired side products, ease of production, etc. A summary of the techniques available are found in several articles, including Steward et. al., Solid Phase Peptide Synthesis, W.H. Freeman, Co. , San Francisco (1969); Bodanszky, et. al., Peptide Synthesis, John Wiley and Sons, Second Edition (1976); J. Meienhofer, Hormonal Proteins and Peptides, 2: 46, Academic Press (1983); Merrifield, Adv. Enzymol. 32: 221-96 (1969); Fields, et. al., Intl. Peptide Protein Res. , 35: 161-214 (1990) and U.S. Patent Number 4,244,946 for solid phase peptide synthesis and Schroder et al , The Peptides, Vol 1 , Academic Press (NY) (1965), for classical solution synthesis. Protecting groups usable in synthesis are described as well in Protective Groups in Organic Chemistry, Plenum Press, New York (1973). Solid phase synthesis methods consist of the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Either the amino or carboxyl group of the first amino acid residue is protected by a suitable selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.

Using a solid phase synthesis method, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is mixed with the solid support and reacted to form an amide linkage with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently to yield the final desired peptide.

The resultant linear peptides may then be reacted to form their corresponding cyclic peptides. A method for cyclizing peptides is described in Zimmer, et.al., Peptides, 393-394 (1992), ESCOM Science Publishers, B.V., 1993. Typically, tertbutoxycarbonyl protected peptide methyl ester is dissolved in methanol and sodium hydroxide solution are added and the mixture treated at 20°C to hydrolytically remove the methyl ester protecting groups. After evaporating the solvent, the tertbutoxycarbonyl protecting groups are then removed under mildly acidic conditions in dioxane cosolvent. The deprotected linear peptide with free amino and carboxy termini is then converted to its corresponding cyclic peptide by reacting a dilute solution of the linear peptide in a mixture of dichloromethane and dimethylformamide, with dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazole and N-methylmorpholine.

The resultant cyclic peptide is then purified by chromatography. Another method to cyclize peptides is described by Gurrath et al, Eur. J. Biochem 210: 911-921 (1992).

To cyclize peptides containing two or more cysteines through the formation of disulfide bonds, the methods described by Tarn, et al, J. Am. Chem. Soc , 113: 6657-6662 (1991); Plaue, Int. J. Peptide Protein Res. , 35: 510-517 (1990); Atherton, J. Chem. Soc. Trans. 1: 2065 (1985); B. Kamber, et. al. Helv. Chim. Acta 63: 899 (1980) are appropriate. Typically, peptides are cyclized in an aqueous, buffered environment having a pH appropriate for cyclization (8-9) at low concentration (0.01 - 10.0 μM). For example, cyclization occurs in 50 mM Tris, pH 8.5 at a concentration of 0.1-1 μM peptide by bubbling C0₂ depleted air through a solution of peptide for 48 hours prior to drying and purification by reverse phase HPLC. This cyclization procedure can be performed before or after cleavage from the solid support. Selective cyclization of cysteine pairs in peptides containing more than two cysteines can be performed using specific protecting groups for desired pairs of cysteines. For example, a combination of two cysteine residues protected with a trityl group (trt) and two cysteine residues protected with an acetamidomethyl group (Acm) in a peptide allows the formation of Cys (trt) disulfide bonds after the initial deprotection of the peptide with trifhioroacetic acid treatment. Subsequently, the Cys (Acm) groups can be deprotected and oxidized at a later stage by a different deprotection and cyclization scheme suitable for the Acm group, such as iodine treatment. Purification of selectively cyclized peptides is then accomplished by reverse phase HPLC as for other peptides.

Peptides may be in the form of any pharmaceutically acceptable salt. Acids capable of forming salts with peptides include inorganic acids such as trifhioroacetic acid (TFA), hydrochloric acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, oxalic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like. Hydrochloric acid and trifluoracetic acid salts are preferred. Suitable bases capable of forming salts with the peptides of the present invention include inorganic bases such as sodium hydroxide and the like as well as organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. , triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine, and the like).

Recomhinant Production

Alternatively, selected compounds of the present invention are produced by expression of recombinant DNA constructs prepared in accordance with well-known methods once the peptides are known. Such production can be desirable to provide large quantities or alternative embodiments of such compounds. Since the peptide sequences are relatively short, recombinant production is facilitated; however, production by recombinant means is preferred over standard solid phase peptide synthesis for peptides of at least 8 amino acid residues. The DNA encoding the sequenced compounds of the present invention is preferably prepared using commercially available nucleic acid synthesis methods. Following these nucleic acid synthesis methods, DNA is isolated in a purified form which encodes the peptides of the invention. Methods to construct expression systems for production of peptides of the invention in recombinant hosts are also generally known in the art. Preferred recombinant expression systems, when transformed into compatible hosts, are capable of expressing the DNA encoding the peptides of the invention. Other preferred methods used to produce peptides of the invention comprise culturing the recombinant host under conditions that are effective to bring about expression of the peptides encoding DNA to produce the peptide of the invention and ultimately recovering the peptides from the culture.

DNA encoding a peptide of this invention, is readily isolated and sequenced using conventional procedures. The recombinant hosts described herein serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transferred into host cells.

Expression can be effected in either procaryotic or eucaryotic hosts. Procaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, such as bacilli, for example Bacillus subtil is, various species of Pseudomonas, or other bacterial strains. In such procaryotic systems, plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used. For example, a workhorse vector for E. coli is pBR322 and its derivatives. Commonly used procaryotic control sequences, which contain promoters for transcription initiation, optionally with an operator, along with ribosome binding-site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems, the tryptophan (trp) promoter system, and the lambda-derived P_L promoter and N-gene ribosome binding site. However, any available promoter system compatible with procaryotes can be used. Expression systems useful in eucaryotic hosts comprise promoters derived from appropriate eucaryotic genes. A class of promoters useful in yeast, for example, includes promoters for synthesis of glycolytic enzymes, e.g. , those for 3- phosphoglycerate kinase. Other yeast promoters include those from the enolase gene or the Leu2 gene obtained from YEpl3.

Suitable mammalian promoters include the early and late promoters from SV40 or other viral promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses. Suitable viral and mammalian enhancers are cited above. In the event plant cells are used as an expression system, the nopaline synthesis promoter, for example, is appropriate.

The expression systems are constructed using well-known restriction and ligation techniques and transformed into appropriate hosts. A preferred expression system would be, when transformed into a compatible host, capable of expressing a DNA encoding a peptide of the invention and inhibiting the binding of the α_vβ₃ integrin receptor with substantially more potency than the natural ligand to the α_vβ₃ integrin receptor. This expression system would comprise peptides of the invention containing the amino acid sequence of formula (I) .

Transformation is done using standard techniques appropriate to such cells. The cells containing the expression systems are cultured under conditions appropriate for production of the peptides, and the peptides are then recovered and purified.

Antibodies

The availability of the purified peptide of the invention also permits the production of antibodies specifically immunoreactive with these forms of the active peptide. The compositions containing purified peptide can be used to stimulate the production of antibodies which immunoreact with the peptides. Standard immunization protocols involving administering peptide to various vertebrates, such as rabbits, rats, mice, sheep, and chickens result in antisera which are immunoreactive with the purified peptide. A peptide may be advantageously conjugated to a suitable antigenically neutral carrier, such as an appropriate serum albumin or keyhole limpet hemocyanin, in order to enhance immunogenicity. In addition, the free peptide can be injected with methylated BSA as an alternative to conjugation. Furthermore, the antibody-secreting cells of the immunized mammal can be immortalized to generate monoclonal antibody panels which can then be screened for reactivity with peptide.

The resulting polyclonal or monoclonal antibody preparations are useful in assays for levels of the corresponding peptide in biological samples using standard immunoassay procedures .

Administration and Utility

Another aspect of this invention is a method for treating a disease that involves the α_vβ₃ integrin receptor by administering a therapeutically effective amount of a peptide of this invention to a patient in need thereof. Generally that amount is sufficient to bind to the α_vβ₃ receptor in a manner that effectively treats and/or prevents the disease. Diseases involving the α_vβ₃ integrin receptor include angiogenic-based diseases (e.g., cancer, anti-inflammatory diseases, retinal neovascular disease, and macular degeneration diseases), diseases related to smooth muscle cell migration (e.g. restenosis) and diseases related to osteoclast-mediated bone resorption (e.g. osteoporosis).

As pointed out, the peptides of the invention are useful therapeutically, to inhibit angiogenesis, i.e. the formation of new blood vessels. Another term for such angiogenesis is neovascularization. Angiogenesis, or neovasculization, includes a variety of processes including "sprouting" , vasculogenesis, or vessel enlargement, all of which are mediated by and dependent upon α_vβ₃ interaction. With the exception of traumatic wound healing, corpus leuteum formation and embryogenesis, it is believed that the majority of angiogenic processes are associated with and/or facilitate a variety of disease processes and states. By inhibiting angiogenesis, therefore, one can intervene in the disease process or state, prevent the disease, ameliorate the signs and symptoms, and in some cases, cure the disease. Compounds of this invention can be referred to as "antineovascularization agents" or "antiangiogenic agents. "

The diseases in which angiogenesis is believed to be important, referred to as angiogenic diseases, include, but are not limited to, inflammatory disorders such as immune and non- immune inflammation, chronic articular rheumatism and psoriasis, rheumatoid arthritis, disorders associated with inappropriate or inopportune invasion of vessels such as diabetic retinopathy, neovascular glaucoma, macular degeneration, capillary proliferation in atherosclerotic plaques and osteoporosis, and cancer associated disorders, such as solid rumors, solid tumor metastases, angiofibromas, skin cancer, retrolental fibroplasia )retinopathy of prematurity), hemangiomas, Kaposi's sarcoma and like cancers that require neovascularization to support tumor growth. To the extent that the compounds of this invention are useful for treating, inhibiting or preventing cancerous tumor growth, they may be referred to as antineoplastic drugs.

The peptides of this invention are also useful therapeutically to treat and/or prevent undesirable smooth muscle cell migration/proliferation. Specific _vβ₃ inhibitors reduce neointima lesion formation. The results of such smooth muscle cell migration are often seen in the occlusion of blood vessels after angioplasty. This process of occlusion of the vessels is referred to as restenosis and is a major problem in patients who have angioplasty performed. The peptides of this invention are also useful therapeutically to treat and/or prevent diseases related to osteoclast-mediated bone resorption. Bone resorption requires the tight attachment of osteoclasts (bone-resorbing cells) to the bone mineralized matrix. Osteoclasts are thought to express α_vβ₃ on their surfaces that interacts with a ligand to provide a signal for the process to go forward. While not wanting to be bound by any theory, the peptides of this invention are thought to bond to _vβ₃ and effectively block the bone resorption process. Thus, the peptides of this invention are useful for treating and/or preventing osteoporosis, a disease of particular importance in an aging population. The use of the compounds of this invention can be used alone or in combination with other therapies that focus on restoring the balance between bone resorption and bone formation (e.g. , the use of estrogens, calcitonin, calcium supplements, vitamin D, bisphosphonates, synthetic PTH analogs, etc.)

The peptides of this invention may be therapeutically useful in other disease states that involve _vβ₃ but that are not specifically disclosed herein but which are apparent to one of ordinary skill upon reading this application in light of other knowledge available in the art. Insofar as that is the case, the treatment of such disease state is meant to be encompassed herein.

In determining whether a peptide of this invention may be useful in the method of this invention one may use any of the available in vitro or in vivo assays available to those skilled in the art. In vitro assays to evaluate the activities of peptide antagonists of the α_vβ₃ integrin receptor include a ligand binding assay using a solid phase ELISA, which is described in the Examples and referenced in Felding- Haberman et al. , J. Biol. Chem. , Vol. 267, pages 5070-5077 (1992) and Orlando, R. A. and Cheresh, D. A, J. Biol. Chem. Vol. 266, pages 19543-19550 (1991). Another in vitro assay includes a cell adhesion assay that shows whether a compound inhibits the ability of α_vβ₃-expressing tumor cells (e.g. M21 melanoma cells) to adhere to vitronectin or fibrinogen. If a compound prevents a cell from adhering, it has the potential to inhibit angiogenesis or metastasis. This is further described in detail in the Examples. See also D. A. Cheresh et al , Cell, 57: 59-69 (1989); N. Kieffer et al. , J. Cell Biol , 113, Number 2: 451-461 (1991); and B. Felding- Haberman et al , J. Clin. Invest. , 89: 2018-2022 (1992). Another in vitro assay contemplated includes the tubular cord formation assay . This in vitro assay shows growth of new blood vessels at the cellular level. See D. S. Grant et al. , Cell, 58: 933-943 (1989) and C. M. Davis et al , J. Cell. Bioc , 51: 206-218.

The peptides of the invention may be tested in certain well-accepted in vivo assays in various test animals such as chickens, rats, mice, rabbits and the like. These in vivo assays include the chicken chorioallantoic membrane (CAM) assay. This assay has been used to show anti-angiogenic activity of both normal and neoplastic tissues. See, D. H. Ausprunk, Amer. J. Path. , 79, No. 3: 597-610 (1975) and L. Ossonowski and E. Reich, Cancer Res. , 30: 2300-2309 (1980). Other in vivo assays include the mouse metastasis assay, which shows the ability of a compound to reduce the rate of growth of transplanted tumors in certain mice or to inhibit the formation of tumors or preneoplastic cells in mice predisposes to cancer or chemically-induced cancer. See M. J. Humphries et al. , Science, 233: 467-470 (1986) and M. J. Humphries et al , J. Clin. Invest. , 81: 782-790 (1988).

While the primary "patient" to whom a compound of this invention is administered will be a human, both male and female, it is to be understood that the compounds of this invention may also be administered to other mammals . These include domestic animals such as dogs, cats and horses as well as livestock such as cows, pigs, goats and the like. Thus the compounds of the invention may be administered by human health professionals as well as veterinarians. In such patients, whether human or otherwise, any variety of tissues or organs can support the disease conditions the peptides of this invention are designed to treat. These tissues and organs include skin, muscle, gut, connective tissue, joints, bones, blood vessels, etc. Another related aspect of the invention is a method for administering a compound of this invention, in conjunction with other therapies such as conventional drug therapy chemotherapy directed against solid tumors and for control of establishment of metastases. The administration of a peptide of this invention is typically conducted during or after chemotherapy, although it is preferable to inhibit angiogenesis after a regimen of chemotherapy at times where the tumor tissue will be responding to the toxic assault. The tumor will attempt to induce angiogenesis to recover by the provision of a blood supply and nutrients to the tumor tissue. Such recovery will be thwarted by the administration of compounds of this invention. In addition, it is preferred to administer a peptide of this invention after surgery where solid tumors have been removed as a prophylaxis against future metastases.

One of ordinary skill will recognize that the potency, and therefore a "therapeutically effective" amount can vary for the compounds of this invention. However, as shown by this specification one skilled in the art can readily assess the potency of a candidate peptide of this invention. Potency can be measured by a variety of means including inhibition of angiogenesis in the CAM assay referred to herein, inhibition of binding of natural ligand to _vβ₃ as described herein, and the like assays. For example a peptide of this invention has the ability to substantially inhibit binding of a natural ligand such as fibrinogen or vitronectin to α_vβ₃ in solution. "Substantial inhibition" is inhibition at an antagonist concentration of less than 10 micromolar (μM), preferably less than 1.0 μM, more preferably less than 0.1 μM, and most preferably less than 0.05 μM. Inhibition is meant that at least a 50 percent reduction in binding of fibrinogen is observed by inhibition in the presence of the vβ₃ antagonist, and at 50% inhibition is referred to herein as an IC₅₀ value.

A unique characteristic of many of the peptides of this invention is that they can exhibit selectivity for _vβ₃ over other integrins. Thus, the preferred peptide antagonist substantially inhibits fibrinogen binding to _vβ₃ but does not substantially inhibit binding of fibrinogen to another integrin, such as _vβ,, α_vβ₅ or _IIbβ₃. Particularly preferred is an _vβ₃ antagonist that exhibits a 10-fold to 100-fold lower IC₅₀ activity at inhibiting fibrinogen binding to α_vβ₃ compared to the IC₅₀ activity at inhibiting fibrinogen binding to another integrin. An exemplary assay for measuring IC₅₀ activity at inhibiting fibrinogen binding to an integrin is described in Example 1.

The peptides of this invention are administered to a patient in need thereof by commonly employed methods for administering peptides in such a way to bring the peptide in contact with the tissue to be treated. Such methods include oral administration, parenteral injection (PI), subcutaneous injection (SC - particularly a controlled release depot), intravenous injection (IV), intramuscularly (IM) and intranasal (IN) or oral inhalation (OI). In general, a therapeutically effective amount is that amount needed to achieve the desired results, i.e. prevent angiogenesis (neovascularization), smooth cell migration (restenosis), or osteoporosis through antagonizing the α_vβ₃ receptor and thus successfully treating of the targeted disease state.

The dosage ranges for the administration of a peptide of this invention (the α_vβ₃ antagonist) depend upon the form of the antagonist, and its potency, as described further herein, and are amounts large enough to produce the desired effect in which the disease symptoms mediated by angiogenesis are ameliorated, e.g. angiogenesis is measurably prevented, inhibited or decreased. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

A therapeutically effective amount of a peptide of this invention is typically an amount of polypeptide such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 nanogram (ng) per milliliter (ml) to about 200 μg/ml, preferably from about 1 ng/ml to about 100 μg/ml. The dosage per body weight can vary from about 0.01 mg/kg to about 3 mg/kg, and preferably from about 0.1 mg/kg to about 1 mg/kg, in one or more dose administrations daily, for one or several days.

The therapeutic compositions containing a peptide of this invention are conventionally administered, whether by PI, IM, IV, INI, SC, or OI, as of a unit dose. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e. , carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect 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 for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but 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 infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

Because of the well-known instability of peptide in the gastrointestinal tract, the compounds of this invention are preferably administered IV, IM, PI, SC, IN or OI. Most preferably the compounds are administered PI using a controlled release delivery or are administered by a noninvasive route. Articles by George Hiller, Advanced Drug-Delivery Reviews, 10: 163-204 (1993) [Elsevier Science Publishers B.V.] and Lorraine L. Wearley, Critical Reviews in Therapeutic Drug Carrier Systems, 8(4): 331-394 (1991) are useful discussions of these types of administrations. These articles are incorporated herein by reference.

Dosage Forms

Another aspect of this invention is a pharmaceutically-acceptable therapeutic composition that comprises a therapeutically effective amount of a peptide of this invention in combination with a pharmaceutically-acceptable excipient. The composition is designed to facilitate the method of administering a peptide of this invention in an effective manner. Generally a composition of this invention will have a peptide dissolved or dispersed in the pharmaceutically - acceptable excipient.

As used herein, the terms "pharmaceutically acceptable" , "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, excipients (including carriers, diluents, stabilizers, lubricants, reagents and the like), are used interchangeably and represent that the materials are capable of administration to or upon a mammal without toxicity and preferably without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

Compounds of this invention may be administered to a mammalian host in a variety of forms depending on the method of administration. The method of administration may be viewed as "invasive" (e.g., IV, IM, PI or SC) or "non- invasive" (e.g., ocular, buccal, oral, transdermal, rectal, NI, OI, [pulmonary], and the like). In general, a composition that is delivered by an invasive route is generally administered by a health care professional while a composition delivered by a non- invasive route may be administered by the patient him- or herself. Compositions that are administered by an invasive route may be designed to be immediate-release or controlled-release over a period of time. Because proteins may be rapidly deactivated by proteolytic enzymes, it is preferable to employ a controlled release dosage form. Nonetheless, immediate release compositions may be used. To aid one of skill the art in determining the peptide stability in different biological media and providing guidance as to the type of delivery system to employ, the following articles are useful: Powell, et al, J. Pharm. Sci. , 81(8): 731-735 (August, 1992) and Powell, et al. , Pharm Res. , 10(9): 1268-1273 (1993).

In administering the compounds of the invention by non-invasive method there are various general methods that are used for enhancing the delivery of proteins. The first is to increase the absorption of the protein. This can be done by the use of a prodrug, chemical modification of the primary structure of the compound, incorporation of the compound into liposomes or other encapsulation material, co- administration with penetration enhancers, the use of physical methods such as iontophoresis and phonophoresis and targeting to specific tissues. Another method for enhancing protein delivery is to minimize the metabolism of the protein. This would include chemical modification of the primary structure, covalent attachment to a polymer, incorporation into a liposome or other encapsulation material, co- administration with an enzyme inhibitor and targeting to specific tissues. The third general method of enhancing protein delivery includes prolonging the half-life of the peptide by protecting it with polymers or liposomes, using a bioadhesive material or targeting the composition to a specific tissue.

In general, if it is desired to increase the absorption of the peptide of this invention through ocular, buccal, transdermal, or rectal administration, or by nasal inhalation or oral inhalation, one can employ certain penetration enhancers. These enhancers can include chelators such as EDTA, citric acid, N-acyl derivatives of collagen, enamines (N- Amino N-acyl derivatives of β-diketones). Surfactants can also be used to enhance penetration. These include, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethelene-20-cetyl ether. Bile salts and derivatives are also known to enhance the penetration of peptides and these include sodium deoxycholate, sodium glycocholate, sodium taurocholate, sodium taurodihydrofusidate and sodium glycodihyrofusidate. Still another type of penetration enhancer useful in the composition of this invention includes ceratin fatty acids and derivatives such as oleic, caprylic acid, capric acid, acylcarnitines, acylcholine and mono and diglycerides. Nonsurfactants are also useful as penetration enhancers. The penetration enhancers can be used in the solution with the compounds of this invention where the compound and the penetration enhancers are in a pharmaceutically acceptable sterile solution which can be administered, for example by nasal administration. Alternatively the penetration enhancers can be included in a powdered formulation that can be administered as an aerosol by suspending the particulate matter in the stream of air and having the patient inhale the suspended particles. Such powdered formulations can be administered by a dry- powder inhaler such as those represented by Ventolin Rotohaler (Glaxo, Inc. ,

Research Triangle Park, North Carolina, U.S. A), and Spinhaler (Fisons Corporation, Bedford, MA). Compositions that are in the form of solid micronized particle having a particle size of about 0.5 to 10 microns in median diameter may be prepared in accordance with the teaching of PCT application international publication numbers WO91/16038 and W093/009 1. Powders may be prepared in accordance with the teaching of Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Chapter 88: 1645-1648. These are in incorporated herein by reference.

The active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1 % of active compound. In preparing oral formulations one needs to be aware of the problems of degradation in the mouth and upper GI tract. Thus, it may be preferable to employ an enzyme inhibitor in combination with the peptide, to use a penetration enhancer or to use a protective polymer or microcapsule. The percentage of the compositions and preparations may, of course, be varied and may conveniently contain up to about 20% by weight of the peptide in a dosage unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 and 50 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain excipients such as the following: a binder such as polyvinylpyrrolidone, gum tragacanth, acacia, sucrose, corn starch, gelatin, calcium phosphate, sodium citrate, and calcium carbonate; a disintegrant such as corn starch, potato starch, tapioca starch, certain complex silicates, alginic acid and the like; a lubricant such as sodium lauryl sulfate, talc and magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin: or a flavoring agent such as peppermint, oil of wintergreen or cherry flavoring. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, flavoring such as cherry or orange flavor, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations. Further components may be apparent to one of ordinary skill in the art.

For purposes of PI administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble, alkali metal or alkaline-earth metal salts. Such aqueous solutions should be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. Solutions of the active compound as a free base or a pharmacologically acceptable salt can be prepared in water suitably mixed with e.g. hydroxypropylcellulose. A dispersion can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. These particular aqueous solutions are especially suitable for IV, IM, SC and PI. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents; for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

For PI formulations that are controlled-release, a peptide of this invention is combined with a polymer that regulates the release of the peptide and protects it from degradation. Generally such polymer may be biodegradable or non-biodegradable and may further be hydrophilic or hydrophobic. Suitable hydrophilic, non-degradable polymers for use in the composition of this invention include hydrogels such as acrylamide or vinyl pyrrolidone crosslinked with N, N' - methylene bisacrylamide. Suitable non-degradable hydrophobic polymers include, ethylene/vinyl acetate copolymers, silicone elastomers, polydimethylsiloxane, and the like. Degradable hydrophilic polymers useful in this invention include N-vinyl pyrrolidone or acrylamide crosslinked with less than 1 % N, N' - methylene bisacrylamide, dextran derivatized with glycidyl methacrylate and crosslinked with N, N' - methylene bisacrylamide, water-soluble polyester prepared from fumaric acid and poly (ethylene glycol) and crosslinked with N-vinyl pyrrolidone, water-soluble polyesters, and the like. Suitable degradable hydrophobic polymers useful for the composition of this invention include lactide/glycolide co-polymers, poly (orthoesters) and poly anhydrides. Of these various polymers, the lactide/glycolide co-polymers are preferred. A more detailed descrPItion of these polymers for controlled parenteral delivery of peptides may be found in an article by George Heller, Advanced Drug- Delivery Reviews, 10: 163-204 (1993) (Elsevier Science Publishers BV). The article is incorporated herein by reference.

For the preferred controlled release composition of this invention the lactide/glycolide co-polymers may have a ratio of DL-a lactic acid to DL-glycolic acid of about 30:70 to about 70:30, preferably about 40:60 to about 60:40. A ratio of about 44:56 is representative. Generally a peptide of this invention is microencapsulated in the copolymer by means known in the art to form the composition, see, for example, U.S. Patent No. 4,675, 189 issued June 23, 1987 to Sanders, Kent, Lewis and Tice. The active peptide is present in an amount from about 0.5% by weight (%w) to about 20.0 %w, preferably about 1.0 %w to about 10.0%w.

For purposes of topical administration, dilute sterile, aqueous solutions (usually in about 0.1 % to 5% concentration), otherwise similar to the above parenteral solutions, are prepared in containers suitable for drop-wise administration to the eye.

The dosage of the present therapeutic agents which will be most suitable for prophylaxis or treatment will vary with the form of administration, the particular compound chosen and the physiological characteristics of the particular patient under treatment. Generally, small dosages will be used initially and, if necessary, will be increased by small increments until the optimum effect under the circumstances is reached. Oral administration requires higher dosages.

It is also contemplated that the peptides of this invention could be delivered to the cells of a subject via gene therapy methods, which are well known in the art. For example, the nucleic acid sequence of each of the peptides of this invention can be determined and such nucleic acids can be synthesized or isolated by standard molecular biology protocols. The nucleic acid encoding the peptide of this invention can be placed into a vector and delivered to the cells of a subject either in vivo or ex vivo by standard methods. The vector can be designed to target cells of the subject which are involved in diseases and pathological conditions associated with the integrin α_vβ₃ receptor and/or the integrin _vβ₅ receptor. Such cell types can include, but are not limited to, cells from skin, muscle, gut, connective tissue, joints, bones and blood vessels. The vector comprising the nucleic acid encoding the peptide of this invention can be delivered to cells under conditions whereby the nucleic acid is expressed and the peptide is produced and secreted from the cell. The production of the peptide can be determined by detecting the presence of the peptide in the cell or in fluid medium associated with the cell (e.g. , culture medium for ex vivo treatment and serum/plasma for in vivo treatment) . The efficacy of administration of the nucleic acid via gene therapy methods with regard to treatment or prevention of a disease or pathological condition can be determined by measuring and monitoring the clinical parameters commonly measured and monitored for a particular disease or condition, as would be well known to the clinician in the art. Improvement in the subject's status as determined by these clinical parameters indicates a successful treatment with the nucleic acids of this invention.

Diagnostic method

Another aspect of this invention is a method for determining the presence of a disease that involves the _vβ₃ integrin receptor. The method comprises

(a) contacting a cell extract from a tissue sample suspected of harboring such disease with measured quantity of a peptide of the present invention that binds α_vβ₃ integrin receptor;

(b) determining the level of binding of the peptide to the _vβ₃ integrin receptor; and (c) comparing the level of binding in (b) to a standard level of binding of said peptide to a cell extract from a healthy tissue sample.

A high level of binding of the peptide to the "diseased" tissue sample indicated the likely presence of an angiogenic-based disease state, an osteoclast-mediated disease state such as osteoporosis or a smooth-muscle cell migration disease state such as restenosis. To determine the level of binding, the peptide is labelled with a detectable label such as a fluorescent or radioactive label. It is to be understood that the compounds preferred for the diagnostic method and kit of this invention are those that are preferred in the same manner as disclosed under the section of this specification entitled "Compounds of the Invention. " It is preferred that a compound that specifically binds α_vβ₃ be used for this method (e.g. , a peptide comprising the binding domain of SEQ ID NO: 3). The diagnostic method of this invention may be of a competitive or non-competitive nature, as those terms are understood in the art.

Another aspect of this invention is a diagnostic kit that includes, in an amount sufficient for at least one assay, a peptide of this invention or a polyclonal antibody or monoclonal antibody of the present invention as a separately packaged reagent. Instructions for use of the packaged reagent are also typically included. Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.

In one embodiments diagnostic system for assaying for the specific presence of the _vβ₃ integrin receptor in a host sample, such as blood, plasma, or tumor cells, comprises a package containing a subject polyclonal antibody that immunoreacts with a polypeptide corresponding to formula (I). In another embodiment, a diagnostic system for assaying for the specific presence of the _vβ₃ integrin receptor in a host sample comprises a package containing a subject monoclonal antibody that unmunoreacts with an epitope formed by the α_vβ₃ integrin receptor-binding region, and preferably also unmunoreacts with a polypeptide of the present invention that binds _vβ₃ integrin receptor A preferred peptide is one that comprises the ammo acid sequence set forth m SEQ ID NO 3 A diagnostic system may also include a subject polypeptide where the assay method to be performed by the diagnostic system utilizes a competitive lmmunoreaction format Further preferred are kits wherein the antibody molecules of the polyclonal or monoclonal antibody are linked to a label

Thus, in preferred embodiments, a diagnostic system of the present invention further includes a label or indicating means capable of signaling the formation of a complex containing the antibody molecules of a polyclonal or monoclonal antibody of the present invention

The word "complex" as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-hgand reaction Exemplary complexes are rmmunoreaction products

As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex "In Vivo" labels or indicating means are those useful within the body of a human subject and include ^{! 11}In, "Tc, ^{Gα 186}Re, and ¹³²I Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents Such labels are themselves well- known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel protein methods and/or systems The linking of labels, i.e., labeling of, polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al. , Meth. Enzymol, 73: 3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al. , Scand. J. Immunol , 8 Suppl. 7: 7-23 (1978); Rodwell et al. , Biotech. , 3: 889-894 (1984) and U.S. Pat. No. 4,493,795.

The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A "specific binding agent" is a molecular entity capable of selectively binding a reagent species of the present invention but is not itself a protein expression product, polypeptide, or antibody molecule of the present invention. Exemplary specific binding agents are antibody molecules, complement proteins or fragments thereof, protein A and the like. Preferably, the specific binding agent can bind the antibody molecule or polypeptide of this invention when it is present as part of a complex.

In preferred embodiments the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.

The diagnostic kits of the present invention can be used in an "ELISA" format to detect the presence or specific quantity of the _vβ₃ integrin receptor in a body fluid sample such as serum, plasma or urine. "ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen or antibody present in a sample. A description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites et al. , published by Lange Medical Publications of Los Altos, CA. in 1982 and in U.S. Pat. No. 3,654,090; U.S.Pat. No. 3,850,752; and U.S. Pat No. 4,016,043, which are all incorporated herein by reference.

Thus, in preferred embodiments, the expressed peptide, or antibody molecule of the present invention can be affixed to a solid matrix to form a solid support that is separately packaged in the subject diagnostic systems.

The reagent is typically affixed to the solid matrix by adsorption from an aqueous medium although other modes of affixation, well known to those skilled in the art can be used.

Useful solid matrices are well known in the art. Such materials include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, N.J.); agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, 111. ; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microliter plate such as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microliter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system. The packages discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. Such packages include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, plastic and plastic-foil laminated envelopes and the like.

The invention now being generally described, the same will be better understood by reference to the following detailed examples, which are provided for the purpose of illustration only and are not to be considered limiting of the invention unless otherwise specified.

EXAMPLES

Example 1 : Solid Phase Integrin α_vβ₃ or _vβ₅ Ligand Binding Assay

This example sets forth a method to evaluate the ability of peptides of this invention to bind to integrin α_vβ₃ or _vβ₅ using a solid phase integrin α_vβ₃ ELISA assay, in which purified integrin α_vβ₃ or α_vβ₅ is presented on the surface of microtiter wells in a native conformation. In this assay, the abilities of peptides in a concentration range extending from 0.01 nM to 1.0 μM to inhibit the binding of biotinylated extracellular matrix protein ligands, e.g. vitronectin or fibronectin, to the integrin α_vβ₃ (Fig. 1A) or α_vβ₅ (Fig. IB) is readily evaluated. Bound ligand is detected with the use of a secondary reagent such as alkaline phosphatase conjugated streptavidin. Comparison of the activities of peptides can be achieved by identifying the concentration at which 50% of the ligand that can bind in the absence of an inhibitor is bound in the presence of the inhibitory peptide. This assay is used to estimate, in a non-physiological setting, the ability of a peptide to bind to α_vβ₃ or α_vβ₅ and can indicate whether a given peptide could treat a disease state that involves the _vβ₃ receptor. The ability of various synthesized peptides to specifically inhibit the binding of vitronectin to α_vβ₃ or _vβ₅ was examined in a microtiter well assay. Immulon 2 plates were coated at 1 μg/ml overnight in coupling buffer (150 mM NaCl , 20 mM Tris pH 7.4, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 0.02% NaN₃), then blocked for 1 hr. with binding buffer containing 3 % BSA. Serial dilutions of peptides in binding buffer (100 mM NaCl, 50 nM Tris pH7.4, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, 0.02 % NaN₃, 0.1 % BSA) were added to wells followed by an equal volume of biotinylated vitronectin at 0.25 μg/ml in binding buffer. The mixture was then incubated for 1 hr. at room temperature and the plates subsequently washed five times with binding buffer followed by a 30 min. incubation with a 1 :2000 dilution of a strepavidin-alkaline phosphatase conjugate. Alkaline phosphatase activity was quantitated using the PMP/AMP detection system (6 mg/ml phenolphthalein monophosphate (PMP) in 0.5 M Tris, pH 10.2, 2% 2-amino-2- methyl-1-propanol (AMP), 0.1 % NaN₃. The color reaction was stopped with an equal volume of 30 mM Tris pH 8.0, 15 mM EDTA and absorbances read at 560 nm. Purified vitronectin was biotinylated with N-hydroxysuccinimidobiotin by mixing 4 μl of a 1 mg/ml stock in DMSO to 40 μg of purified vitronectin in PBS for 2 hrs. at room temperature. The biotinylated protein was then dialyzed against PBS using a 50 kD molecular weight cut off.

Example 2: Cell Adhesion Assay

This example sets forth a method to evaluate the ability of a peptide of this invention to inhibit the ability of α_vβ₃-expressfng melanoma cells to adhere to ligands.

In this assay, various concentrations of peptides are incubated with freshly resuspended cells from cell culture which are plated onto extracellular matrix proteins in the presence of the divalent cation co-factors required for integrin function. The IC50, or concentration of peptides required to inhibit 50% of the degree of adhesion exhibited by cells incubated on extracellular matrix proteins in the absence of inhibitors, can be used to compare the effectiveness of inhibitory reagents.

96 well Immulon 2 plates were coated with 100 μl of 10 μg/ml fibrinogen (Calbiochem, La Jolla, CA) in PBS overnight at 4°C. Plates were then blocked for 1 hr. at 37°C with 3 % BSA in PBS. Serial dilutions of peptides resuspended in adhesion buffer (IX HBSS supplemented with 10 mM HEPES pH 7.4, 0.2 mM MnCl₂, 1 % BSA, and an additional 2 mM MgCl₂ and 2 mM CaCl₂) were then added in 100 ul aliquots to the fibrinogen coated wells for 30 min. at 37°C. M21 melanoma cells, which express high levels of the _vβ₃ integrin, were grown in RPMI supplemented with 2mM glutamine and 10% FBS. Cells were trypsinized briefly and washed twice with 37°C adhesion buffer before resuspending at 4xl0⁵ cells/ml. 100 μl of this cell suspension was then added to each peptide dilution and the plates incubated for 20 mins. at 37° C to allow cells to settle and adhere to fibrinogen. Plates were gently washed 4 times with adhesion buffer to remove non-adherent cells and bound cells detected by staining with 100 μl of 0.5 % crystal violet in 20% methanol for 30 min. Excess stain was removed by extensvie washing with deionized water. Stained cells were solublized in 100 μl 10% acetic acid, transfered to another 96 well plate, and the absorbance read at 560 nm.

The IC₅₀ peptide concentration is determined from the amount of peptide required to inhibit 50% of the binding exhibited by cells in the absence of peptide or other antagonists after background binding on BSA was subtracted. A particularly desirable peptide is one with an activity of 20 μM or below.

Example 3: Cell Migration Assay

This example describes a method to evaluate the ability of peptide of this invention to inhibit cytokine-induced smooth muscle cell migration. Smooth muscle cell migration is a prerequisite for restenosis, and the assay identifies peptide inhibitors of this migratory process, and thus, potential inhibitors of restenosis. In this assay the number of cells that migrate in response to cytokine induction and the ability of various peptides to inhibit is easily measured. See also variations of this assay in Choi, E.T. et al , J. Vase. Surg. , Vol. 19, pages 125-134 (1994).

Supplies: Costar Trans well 8.0 μm (Catalog #3422)

PDGF, bFGF or other chemoattractant

Vitronectin, fibrinogen or other extracellular matrix ligand

BSA Serum-free medium, supplemented only with insulin, antibiotics, 10 mM HEPES, pH 7.4, 2 mM CaCl₂, 2 mM MgC12

Day O: Passage cells 2:3 to obtain healthy, log-phase cultures.

Day 1 : Dilute chemoattractant (PDGF, bFGF, etc., 1-10 ng/ml) and matrix protein (1-20 μg/ml) in insulin and antibiotic supplemented growth medium. Include controls for non-specific migration (0.5 % BSA) and random migration (matrix protein in lower and upper chambers). Aliquot 500 μl into lower chamber, allow to equilibrate at 37°C, while preparing cells. This allows matrix protein to coat the lower surface.

Harvest cells: Wash monolayer with PBS or other saline solution. Treat with trypsin/EDTA, 5 ml/25 cm² of surface area for 5 min. Add BSA-containing migration buffer, triturate to obtain single cell suspension, count an aliquot in hemocytometer. Wash cells by centrifugation and resuspension to remove serum. Resuspend at 5 x 10⁵ cells per ml in migration buffer plus inhibitors.

Add 100 μl to upper chamber. Incubate 37 oC, 5% C0₂ for 6-18 hours, depending on cell type. ATTORNEY DOCKET NO. 09051.0004/P

Day 2: Recover membrane insert. Remove cells from inner portion with Q-

Tip cotton swab and place into a second tray containing 250 μl freshly prepared 1 % crystal violet in 20% methanol, 80% water. Stain for 20 minutes.

Remove insert and wash thoroughly in water. Drain and dry the upper membrane surface with cotton swab. Oven dry until absolutely dry.

Extract the dye by placing dried insert into clean tray containing 250 μl 10% acetic acid. Extract for 5 minutes and agitate to ensure complete extraction. Discard membrane insert, recover an aliquot of dye extract and read at 500 nm.

Example 4: Screening Peptide Libraries Using Phage Display

This example provides a method for preparing peptide libraries that can be used for screening and optimizing peptides for a specific receptor generally in accordance with the method of Scott, J. K. and Smith, G. P. Science, 249: 386-390 (1990). A phage display library comprises tens to hundreds of millions of variable amino acid sequences that are displayed on the surface of a bacteriophage virion. Generally, two viral proteins have been used to display peptides, a minor coat protein pill and the major coat protein pVIII. Both proteins are synthesized with short signal sequences that allow them to be transported to the bacterial inner membrane, where they are cleaved by signal peptidase to form mature proteins. The mature proteins are added to the viral coat as it assembles in and emerges from the inner membrane. The major coat protein, pVIII, forms the body of the phage, whereas four or five copies of the minor coat protein, pill, are added at the trailing end of the merging virion. In both coat proteins, the peptide is inserted at or within a few residues of the amino terminus of the mature protein. To make a phage library, a degenerate oligonucleotide is synthesized using single nucleotides at positions encoding the invariant amino acids and variable nucleotides at positions encoding variable amino acids. For example, all 20 amino acids may be represented by NNX where N represents the equimolar amounts of the four nucleotides and X can either be equimolar amounts of G and T or equimolar amounts of G and C. The appropriate ends for ligation with the vector can then be generated by restriction endonuclease digestion. After the ligated product is introduced into bacterial cells by electroporation, the phage are propagated, collected, and purified. Very large libraries of up to 200-300 million independent clones have been constructed using the phage method.

Synthesis of oligonucleotides

All oligonucleotides were synthesized on an Applied Biosystems Model 394 DNA/RNA synthesizer (Applied Biosystems, Foster city, CA) using standard phosphoramidate chemistry. Controlled pore glass (CPG) beads were used as the synthesis support for oligonucleotides for standard mutagenesis and polystyrene beads (ABI) for oligonucleotides for library construction.

The synthesis of N-terminal oligonucleotides was started by packing 1.0 μmol of dimethoxytrityl derivatized deoxyribonucleotide polystyrene beads into each of ten separate 1.0 μmol synthesis columns. Since the synthesis proceeds in the 3' to 5' direction, the 3' complementary end and the first random triplet were initially synthesized on the columns. Following this, the ten columns were opened and the beads were emptied into a vessel and 2 ml of acetonitrile was added. The suspension was mixed by pipetting up and down and 200 μl was aliquotted into ten new columns. After capping the columns, the next triplet was synthesized. The columns were once again opened, mixed and divided into ten new columns. This was repeated ten times for the randomized libraries and nine times for the cysteine enriched libraries as well as for the non-RGD library. After synthesis of the last random triplet, the beads were again mixed into 2 ml acetonitrile and the suspension was equally divided into forty individual columns by pipetting fifty μl into each. The 5' ends of the oligonucleotides were synthesized in the same fashion but using different sequences. The forty N- and forty C- terminal oligonucleotides were purified by OPC columns (ABI) or by denaturing gel electrophoresis.

Four libraries were constructed which were enriched in cysteine residues and excluded a carboxyl terminal glycine after arginine. Separate columns were now included for glycine and arginine and an extra column was included for cysteine. Since cysteine was encoded by the same triplet combination as arginine in the fully random synthesis and tryptophan on the glycine column, cysteine was now combined with tryptophan. This synthesis was performed using 0.2 μmol of the polystyrene beads per column except for the glycine and arginine columns which received 0.1 μmol beads. Following the synthesis of the 3' complimentary end and the first triplet, columns 2-12, were opened and the resins mixed into 2.1 ml acetonitrile. One hundred μl was added into a new column number 1 for the synthesis of a triplet coding for arginine and 200 μl was aliquotted. This procedure was repeated nine times as described above and the 5' ends of the oligonucleotides were then added. Two oligonucleotides were synthesized to make libraries with a cysteine as residue 10 and two with an alanine as the tenth residue. The carboxyl terminal oligonucleotide was essentially made in a similar way, except that the mixing order for columns 1 and 2 was reversed. This is because the carboxyl terminal oligonucleotide is the reverse complement of the amino terminal. Thus, after the mixing of columns 1 and 3-12, one hundred μl was packed into a new column number 1 for the synthesis of a triplet coding for arginine. Column number 2 was then added to the remaining suspension as for the amino terminal oligonucleotide. Vectors

The vectors were constructed by sequential stepwise oligonucleotide-directed in vitro mutagenesis (Kunkel, 1987). Both the N-terminal, ED07 and the C-terminal, 1X47 vectors were derived from the previously described ED03 and 1X421 vectors. They are originally engineered from M13 mpl9 (Yanisch-Perron et al. , 1985) and contain all necessary elements for the normal growth of this filamentous phage.

ED07 which is used for the construction of the N-terminal half of the peptide libraries is essentially similar to ED03 except that the Fokl site at position 3547 was eliminated. Bsal sites at positions 6366 and 6866 were introduced as well as two EcoRl sites at positions 6386 and 6852.

1X47 was constructed from 1X40 which is a precursor of 1X421. 1X40 is identical to 1X421 except for a change in the Fokl overhang at position 3547 (TTTT rather than CTTC) and a T rather than a C at position 4492. These differences have no relevance in the present work and are mentioned here only for the completeness of vector description. A Xbal and a Xhol site were introduced into 1X40 at positions 5877 and 6748, respectively. The vector was cut with these enzymes and the larger fragment was saved. Next, Xhol and Xbal sites were introduced into the original 1X40 at positions 5876 and 6744, respectively. Restriction digestion with these enzymes released a 868 nucleotide long fragment which was ligated into the complementary ends of the large fragment. This construct was saved as the C- terminal vector 1X47.

Construction of Peptide Libraries

The libraries are built in two steps. In the first step, separate N- and C- terminal libraries (sub-libraries) are made which are subsequently joined into the combinatorial library. Uracil containing single-stranded ED07 and 1X47 DNA was

Claims

isolated. To obtain enough material, two identical mutagenesis reactions were then performed to introduce the random oligonucleotides into each vector. Each reaction contained 2.5 pmol uracil DNA, 125 pmol oligonucleotide (molar ratio of template oligo is 1:5), 20 mM Tris-HCL, pH 7.4, 2 mM MgCl, and 50 mM NaCl in a total volume of 50 μl. The reactions were heated to 75° C for 2 minutes and cooled to room temperature over a 40 minute period for annealing of the oligonucleotides to the vector. After annealing, the reactions were transferred to ice and 15 μl of a buffer containing 5 mM of each dATP, dCTP, dGTP, and dTTP, 10 mM ATP, lOOmM Tris-HCL, pH 7.4, 50 mM MgCl2, 20 mM DTT, 5 U T7 DNA polymerase and 20 U T4 DNA ligase was added. The reactions were kept on ice for 5 minutes followed by incubation at room temperature for 5 minutes and 37° C for 1 hour. The two identical reactions were pooled, extracted with phenol/CHCl3, precipitated with ethanol and resuspended in 21 μl of TE pH 8.0. Multiple electroporations were performed into DH10B cells to obtain at least 108 transformants in each sub-library. Usually 4 transformations, each with 2 μl of the mutagenesis reaction was enough to give the required number of clones. Each set of electroporations was added into 6 ml of 2x YT media and incubated while shaking at 37° C for 8 hours. The phage containing supernatant was saved as the amplified sub-library.In the second step of the library construction, RF DNA from each sub-library was prepared. One hundred μg of each sub-library DNA was cut to completion with Bsal, extracted with phenol/CHCl3 and resuspended in 100 μl of TE pH 7.4. The digestion of both the N- and C-terminal plasmids gives rise to one large (approximately 6.5 kb) and one small (approximately 0.5 kb) fragment. The N- terminal large fragment is ligated with the C-terminal small fragment to generate the combinatorial library. The desired fragments were isolated on a Gen-Pak FAX (Waters) HPLC column at 30° C. The sample (100 μl) was injected onto a column equilibrated with 60% buffer A (25 mM Tris-HCl, pH 8.0, lmM EDTA) and 40% buffer B (25 mM Tris-HCL, pH 8.0, 1 mM EDTA, IM NaCl) using a flow rate of 0.5 ml/min. A linear gradient of 40% buffer A, 60% buffer B was run over 40 ATTORNEY DOCKET NO. 09051.0004/Pminutes. In this system, the smaller fragment eluted after approximately 30 minutes followed by the larger piece at about 40 minutes. Fractions containing the large fragment (6.5 kb) from the N-terminal library and the small fragment from the C- terminal library were collected and pooled. Both fragments were recovered by centrifugation after the addition of an equal volume of isopropanol and keeping them on ice for 30 minutes. This procedure eliminated the co-precipitation of NaCl with the DNA. Each fragment was resuspended in 50 μl TE pH 7.4.Three ligation reactions were set up, each containing 10 μl of both the large and small fragment, 20 mM Tris-HCL pH 7.6, 5 mM MgCL, 0.5 mM DTT and 1 mM ATP in a final volumn of 400 μl. Ligations were carried out at 15° C for at least 16 hours. The three ligations were pooled, extracted with phenol/CHCl3 and precipitated twice with ethanol before resuspension in 30 μl TE pH 8.0. Multiple electrotransformations were performed to obtain at least IxlO9 primary transformants. Usually, ten transformations, each with 2 μl DNA and 35 μl competent cells were enough to generate the desired amount of transformants. The library was then amplified using standard protocols.Preparation of Electroco petent CellsElectrocompetent XL-1 Blue, DH10B, or MC 1061 cells were prepared by inoculating 500 ml 2x YT media in a 2 liter baffle flask with several loopfulls of bacterial colonies that were grown overnight on LB plates. The culture was incubated at 37° C in an orbital shaker until late log phase. The culture was chilled on wet ice and the bacteria were pelleted at 5000xg for 15 minutes at 4° C. The pellet was resuspended in 500 ml ice cold deionized water and centrifuged again. The previous step was repeated once using 250 ml water. The pellet was then resuspended in 100 ml 10% glycerol in water and centrifuged. The final pellet was resuspended in 1-2 ml residual supernatant, frozen in aliquots in an ethanol/dry ice bath and stored a -80° C. Care was taken throughout the procedure to keep the bacteria at 4° C.Isolation of the replicative form of M13 phageFive ml of overnight XL-1 Blue cells were added to 500 ml 2x YT media. After incubation for 1.5 hour, 10 μl of a high titer phage stock (1011 pfu/ml) were added and the culture was incubated for an additional 6 hours. Bacteria were harvested by centrifugation and washed once to decrease contamination of the plasmid with single-stranded phage DNA. The cells were resuspended in 20 ml 10 mM EDTA, pH 8.0 and 40 ml 0.2 M NaOH, 1 % SDS was added. After 10 minutes on ice, 20 ml of 5 M potassium acetate was added. The mixture was shaken and incubated on ice for 5 minutes. After centrifugation at 7000xg for 15 minutes, the supernatant was filtered through a single layer of a Kimwipe tissue and an equal volume of isopropanol was added to precipitate the nucleic acids. The pellet was resuspended in 10 ml of 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 and 50 μl of 10 mg/ml RNase A was added. After incubation for 15 minutes at 37° C, the mixture was extracted with phenol/CHCl3 and precipitated with ethanol. The pellet was resuspended in 2 ml of 10 mM Tris-HCL, 1 mM EDTA pH 8.0, and the plasmid DNA was precipitated with polyethylene glycol before final purification by centrifugation on a CsCl/EtBr gradient.VectorsThe N-terminal (ED03) and C-terminal (1X421) peptide library vectors were constructed by stepwise sequential oligonucleotide-directed in vitro mutagenesis. Both vectors are derived from M13 mpl9.EDJ03 was constructed in short as follows: A pseudo-wild type gene VIII sequence was synthesized by annealing overlapping oligonucleotides. The oligonucleotides were phosphorylated and heated to 65 °C. After slow cooling to room temperature the annealed product was ligated into dephosphorylated M13 mpl9 cut with EcoRl and Hindlll. These restriction sites were subsequently destroyed in the final vector construct by mutagenesis. The lac Z gene was deleted and Fokl restriction enzyme sites at positions 239 and 7244 were removed. The Fokl overhang at position 3561 was changed from TTTT to CTTC. Overlapping oligonucleotides were used to construct a phosphorylase A leader sequence, in which amino acid at position 19, coding for alanine, was changed to encode threonine. This sequence was inserted between the lac promoter/ operator and pseudo-gene VIII. Finally, 33 nucleotides encoding the sequence YGGFMLLRHPG, was inserted between the leader sequence and pseudo gene VIII. All nucleotide changes within coding sequences of the phage genome were made without changing the coding information.The vector 1X421 was constructed by first synthesizing the pseudo gene VIII sequence with BamHl and Hindlll ends. It was then ligated into M13 mpl9, cut with the same enzymes, and dephosphorylated. The lac Z gene was deleted and Fokl sites at positions 239 and 7244 were removed and the Fokl overhang at nucleotide 3561 was changed from TTTT to CTTC in analogy with the construction of EDJ03. A Fokl site was introduced at position 6223.Example 5: ELISA-based Phage Binding AssayThe ability of various peptide-expressing phage to bind to vβ3 and vβ5 was evaluated using an ELISA-based assay. Immulon 2 plates are coated overnight at 4° C with 1 μg/ml αvβ3 in coupling buffer (150 mM NaCl, 20 mM Tris ph 7.4, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.02% NaN3). Plates are blocked for 2 hr with binding buffer 100 mM NaCl, 50 mM NAC1, 50 mM Tris ph 7.4, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.02% NaN3, 1 mg/ml BSA (0.1 % BSA)) lacking BSA, but containing 5% nonfat dried milk instead. Plates are then washed 3 times with binding buffer to remove any residual nonfat dried milk before the addition of any phage. High titer peptide-expressing phage in Luria Broth supernatants, or PEG precipitated purified phage resuspended in binding buffer, are then added to plates and incubated for 1 hr at room temperature. Bound phage are detected by inhibiting for 1 hr at room temperature (lOOmg/ml in binding buffer) a biotinylated rabbit anti- M13 antibody followed by a strepavidin alkaline phosphatase conjugate (1 :4000 dilution in binding buffer). Alkaline phosphatase activity is determined using phenolphthalein monophosphate (PMP) as a substrate and absorbances measured at 560mM. Between the addition of each reagent, plates are washed a total of 6 times with binding buffer.All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.The invention now being fully described, it will be apparent to one of oridinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims . SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: IXSYS , INC. (ii) TITLE OF THE INVENTION: PEPTIDE INHIBITORS OF α„β3 and vβ5 (iii) NUMBER OF SEQUENCES: 32 (iv) CORRESPONDENCE ADDRESS:(A ADDRESSEE: NEEDLE & ROSENBERG, P.C. (B STREET: 127 PEACHTREE STREET, NE, SUITE 1200 (C CITY: ATLANTA (D STATE : GA (E COUNTRY : USA (F ZIP: 30303-1811(v) COMPUTER READABLE FORM:(A MEDIUM TYPE: Diskette (B COMPUTER: IBM Compatible (C OPERATING SYSTEM: DOS (D SOFTWARE: FastSEQ for Windows Version 2.0(vi) CURRENT APPLICATION DATA: (A APPLICATION NUMBER: (B FILING DATE: 15-JUN-1998 (C, CLASSIFICATION:(vii PRIOR APPLICATION DATA: (A APPLICATION NUMBER: U.S. Serial No. 08/482,106; 08/482,107(B : FILING DATE: 6/7/95; 6/7/95 viii) ATTORNEY/ GENT INFORMATION:(A NAME: Perryman, David G (B REGISTRATION NUMBER: 33,438 (C REFERENCE/DOCKET NUMBER: 09051.0004/P(ix) TELECOMMUNICATION INFORMATION : (A TELEPHONE: 404 688 0770 (B TELEFAX: 404 688 9880 (C TELEX :(2) INFORMATION FOR SEQ ID NO : 1 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(C) STRANDEDNESS : unknown(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide (ix) FEATURE: (A NAME/KEY: Other(B LOCATION: 1(D OTHER INFORMATION: Xaa = Any number of ammo acids, from-11, at the N-termmus .(A NAME/KEY: Other (B LOCATION : 2 (D OTHER INFORMATION. Xaa = any ammo acid(A. NAME/KEY: None(B LOCATION: 4 (D OTHER INFORMATION Xaa any ammo acid(A NAME/KEY. None(B LOCATION: 5(D OTHER INFORMATION Xaa = Pro or Cys (which moiety may be protected or unprotected)(A NAME/KEY. None (B LOCATION: 7 (D OTHER INFORMATION. Xaa = Ala, Ser, Thr, Asn, Leu, Gin, or Asp(A NAME/KEY: None (B LOCATION: 10 (D OTHER INFORMATION- Xaa = any ammo acid(A NAME/KEY: None (B LOCATION. 11 (D OTHER INFORMATION. Xaa = any number of ammo acids, from 0- 11, at the C-terminus(xi) SEQUENCE DESCRIPTION. SEQ ID NO : 1Xaa Xaa Cys Xaa Xaa Arg Xaa Asp Cys Xaa Xaa1 5 10(2) INFORMATION FOR SEQ ID NO : 2(l) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 ammo acids (C) STRANDEDNESS : single(D) TOPOLOGY: linear(n) MOLECULE TYPE: None(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :Thr Cys Glu Cys Arg Ala Asp Cys Tyr Cys1 5 10(2) INFORMATION FOR SEQ ID NO : 3 :(l) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 ammo acids (C) STRANDEDNESS : single(D) TOPOLOGY: linear(11) MOLECULE TYPE. None(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3Thr Cys Ser Pro Arg Ala Asp Cys Ala1 5(2) INFORMATION FOR SEQ ID NO : 4 :(l) SEQUENCE CHARACTERISTICS:(A) LENGTH. 9 ammo acids(B) TYPE, ammo acid(C) STRANDEDNESS single(D) TOPOLOGY linear(n) MOLECULE TYPE None(xi) SEQUENCE DESCRIPTION: SEQ ID NO 4Arg Cys Gly Gly Asp Ser Met Cys Tyr 1 5(2) INFORMATION FOR SEQ ID NO : 5 :(l) SEQUENCE CHARACTERISTICS.(A) LENGTH 9 ammo acids(B) TYPE, ammo acid(C) STRANDEDNESS single(D) TOPOLOGY- linear(ii) MOLECULE TYPE. None(xi) SEQUENCE DESCRIPTION SEQ ID NO : 5Arg Cys Gly Gly Asp Ser Asp Cys Tyr1 5(2) INFORMATION FOR SEQ ID NO : 6 :(I) SEQUENCE CHARACTERISTICS:(A) LENGTH- 10 ammo acids (C) STRANDEDNESS. single(D) TOPOLOGY- linear(II) MOLECULE TYPE: None (IX) FEATURE:(A) NAME/KEY. Other(B) LOCATION: 2(D) OTHER INFORMATION: Xaa = pencillamme(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6. Gly Xaa Gly Arg Gly Asp Ser Pro Cys Ala1 5 10(2) INFORMATION FOR SEQ ID NO : 7 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None( i) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :Thr Cys Ser Pro Arg Ala Glu Cys Ala Ala 1 5 10(2) INFORMATION FOR SEQ ID NO : 8 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: ammo acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 3 & 4(D) OTHER INFORMATION: Xaa = any amino acid(A) NAME/KEY: Other(B) LOCATION: 9(D) OTHER INFORMATION: Xaa = any ammo acid(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :Thr Cys Xaa Xaa Arg Ala Asp Cys Xaa 1 5(2) INFORMATION FOR SEQ ID NO : 9 :(l) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other (B) LOCATION: 7(D) OTHER INFORMATION: Xaa = any amino acid(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :Arg Cys Gly Gly Asp Ser Xaa Cys Tyr 1 5(2) INFORMATION FOR SEQ ID NO: 10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 5 amino acids(B) TYPE: ammo acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:Gly Arg Gly Asp Ser1 5(2) INFORMATION FOR SEQ ID NO: 11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: ammo acid(C) STRANDEDNESS: unknown(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 2(D) OTHER INFORMATION: Xaa = pencillamme(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:Gly Xaa Gly Arg Gly Asp Ser Pro Cys Ala1 5(2) INFORMATION FOR SEQ ID NO: 12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12 :Gly Arg Gly Asp Ser Pro1 5 (2) INFORMATION FOR SEQ ID NO : 13 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 ammo acids(B) TYPE: ammo acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 13 :Ala Cys Pro Ser Arg Leu Asp Ser Pro Cys Gly 1 5 10(2) INFORMATION FOR SEQ ID NO : 14 :(l) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 ammo acids(B) TYPE: ammo acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 5(D) OTHER INFORMATION: Xaa = any amino acid(A) NAME/KEY: Other(B) LOCATION: 7(D) OTHER INFORMATION: Xaa = any ammo ac d(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 14 :Arg Cys Asp Gly Xaa Cys Xaa1 5(2) INFORMATION FOR SEQ ID NO: 15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 3(D) OTHER INFORMATION: Xaa = Gly, Ala, or Ser (A) NAME/KEY: Other(B) LOCATION: 6 & 7(D) OTHER INFORMATION: Xaa = any amino acid(A) NAME/KEY: Other(B) LOCATION: 9(D) OTHER INFORMATION: Xaa = any amino acid(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:Arg Cys Xaa Gly Asp Xaa Xaa Cys Xaa 1 5INFORMATION FOR SEQ ID NO: 16(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:Thr Cys Ser Pro Arg Xaa Asp Cys Ala 1 5(2) INFORMATION FOR SEQ ID NO: 17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Thr Cys Ser Pro Arg Xaa Asp Cys Ser 1 5(2) INFORMATION FOR SEQ ID NO: 18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:Thr Cys Ser Pro Arg Xaa Asp Cys Tyr 1 5(2) INFORMATION FOR SEQ ID NO: 19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:Tyr Cys Ser Pro Arg Xaa Asp Cys Ser1 5(2) INFORMATION FOR SEQ ID NO: 20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other ATTORNEY DOCKET NO. 09051.0004/P(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:Thr Cys Glu Pro Arg Xaa Asp Cys Ser 1 5(2) INFORMATION FOR SEQ ID NO: 21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:Thr Cys Glu Pro Arg Xaa Asp Cys Tyr 1 5(2) INFORMATION FOR SEQ ID NO: 22:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:Thr Cys Glu Pro Arg Xaa Asp Cys Ala 1 5(2) INFORMATION FOR SEQ ID NO: 23:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid ATTORNEY DOCKET NO. 09051.0004/P(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4(D) OTHER INFORMATION: Cys may be protected or unprotected(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(A) NAME/KEY: Other(B) LOCATION: 10(D) OTHER INFORMATION: Cys may be protected or unprotected(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:Thr Cys Ser Cys Arg Xaa Asp Cys Ala Cys 1 5 10(2) INFORMATION FOR SEQ ID NO: 24:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 ammo acids(B) TYPE: ammo acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4(D) OTHER INFORMATION: Cys may be protected or unprotected(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(A) NAME/KEY: Other(B) LOCATION: 10(D) OTHER INFORMATION: Cys may be protected or unprotected(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24 ATTORNEY DOCKET NO. 09051.0004/PThr Cys Ser Cys Arg Xaa Asp Cys Ser Cys 1 5 10(2) INFORMATION FOR SEQ ID NO: 25:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4(D) OTHER INFORMATION: Cys may be protected or unprotected(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(A) NAME/KEY: Other(B) LOCATION: 10(D) OTHER INFORMATION: Cys may be protected or unprotected(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:Tyr Cys Ser Cys Arg Xaa Asp Cys Ala Cys1 5 10(2) INFORMATION FOR SEQ ID NO: 26:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4(D) OTHER INFORMATION: Cys may be protected or unprotected(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(A) NAME/KEY: Other (B) LOCATION: 10(D) OTHER INFORMATION: Cys may be protected or unprotected(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:Thr Cys Ser Cys Arg Xaa Asp Cys Tyr Cys1 5 10(2) INFORMATION FOR SEQ ID NO: 27:(l) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 ammo acids (C) STRANDEDNESS. single(D) TOPOLOGY: linear(ii) MOLECULE TYPE. None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4(D) OTHER INFORMATION: Cys may be protected or unprotected(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION- Xaa = Ala, Ser, Thr, or Leu(A) NAME/KEY: Other(B) LOCATION: 10(D) OTHER INFORMATION Cys may be protected or unprotected(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:Tyr Cys Ser Cys Arg Xaa Asp Cys Tyr Cys 1 5 10(2) INFORMATION FOR SEQ ID NO: 28:(I) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 ammo acids(B) TYPE: ammo acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(II) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4(D) OTHER INFORMATION: Cys may be protected or unprotected (A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(A) NAME/KEY: Other(B) LOCATION: 10(D) OTHER INFORMATION: Cys may be protected or unprotected(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:Tyr Cys Ser Cys Arg Xaa Asp Cys Ser Cys 1 5 10(2) INFORMATION FOR SEQ ID NO: 29:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 4(D) OTHER INFORMATION: Cys may be protected or unprotected(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = Ala, Ser, Thr, or Leu(A) NAME/KEY: Other(B) LOCATION: 10(D) OTHER INFORMATION: Cys may be protected or unprotected(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:Thr Cys Glu Cys Arg Xaa Asp Cys Tyr Cys 1 5 10(2) INFORMATION FOR SEQ ID NO: 30:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None ( ix) FEATURE :(A) NAME/KEY: Other(B) LOCATION: 1(D) OTHER INFORMATION: Xaa = Any number of amino acids, from 0-2, at the N-Terminus.(A) NAME/KEY: Other(B) LOCATION: 6(D) OTHER INFORMATION: Xaa = any amino acid(A) NAME/KEY: Other(B) LOCATION: 8(D) OTHER INFORMATION: Xaa = any number of amino acids, from 0-3, at the C-terminus.(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:Xaa Arg Cys Asp Gly Xaa Cys Xaa 1 5(2) INFORMATION FOR SEQ ID NO : 31 :(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other(B) LOCATION: 7(D) OTHER INFORMATION: Xaa = any amino acid(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:Arg Cys Asp Gly Pro Cys Xaa 1 5(2) INFORMATION FOR SEQ ID NO: 32:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: None (ix) FEATURE:(A) NAME/KEY: Other (B) LOCATION: 7(D) OTHER INFORMATION: Xaa = any amino acid(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:Arg Cys Asp Gly lie Cys Xaa 1 5 What is claimed is:

1. An amino acid sequence comprising the binding domain Thr-Cys-X₁-X₂-Arg- Ala-Asp-Cys-X₃ (SEQ ID NO: 8) which binds integrin α_vβ₃.

2. The amino acid sequence of claim 1, comprising nine to thirty amino acid residues

3. The amino acid sequence of claim 1, wherein the binding domain contains a cyclic portion formed by a disulfide bond between two Cys residues in the binding domain.

4. The amino acid sequence of claim 1, wherein X, is Glu.

5. The amino acid sequence of claim 1, wherein X₂ is Cys.

6. The amino acid sequence of claim 1, wherein X₃ is Tyr.

7. The amino acid sequence of claim 1, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

8. The amino acid sequence of claim 1, wherein X, is Ser.

9. The amino acid sequence of claim 1, wherein X₂ is Pro.

10. The amino acid sequence of claim 1, wherein X₃ is Ala.

11. The amino acid sequence of claim 1 , wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:3.

12. An amino acid sequence comprising the binding domain Arg-Cys-Gly-Gly- Asp-Ser-X₄-Cys-Tyr (SEQ ID NO: 9) which binds integrin _vβ₃.

13. The amino acid sequence of claim 12, comprising nine to thirty amino acid residues.

14. The amino acid sequence of claim 12, wherein the binding domain contains a cyclic portion formed by a disulfide bond between two Cys residues in the binding domain.

15. The amino acid sequence of claim 12, wherein X₄ is Met.

16. The amino acid sequence of claim 12, wherein X₄ is Asp.

17. The amino acid sequence of claim 12, wherein the amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO:4.

18. The amino acid sequence of claim 12, wherein the amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 5.

19. A peptide mimetic of the amino acid sequence of claim 1, 7 or 11.

20. A peptide mimetic of the amino acid sequence of claim 12, 17 or 18.

21. A method of inhibiting binding of a ligand to integrin αvβ3 expressed on a cell in a subject comprising administering to the cell the amino acid sequence of claim 1.

22. The method of claim 21, wherein the amino acid sequence comprises the amino acid sequence as set forth in SEQ ID NO: 2.

23. The method of claim 21, wherein the amino acid sequence comprises the amino acid sequence as set forth in SEQ ID NO: 3.

24. A method of inhibiting binding of a ligand to integrin α_vβ₅ expressed on a cell in a subject comprising administering to the cell the amino acid sequence of claim 7.

25. A method of inhibiting binding of a ligand to integrin α_vβ₃ expressed on a cell in a subject comprising administering to the subject the amino acid sequence of claim 12.

26. The method of claim 25, wherein the amino acid sequence has the amino acid sequence as set forth in SEQ ID NO: 4.

27. The method of claim 25, wherein the amino acid sequence has the amino acid sequence as set forth in SEQ ID NO: 5.

28. A method of inhibiting binding of a ligand to integrin α_vβ₅ expressed on a cell in a subject comprising administering to the subject the amino acid sequence of claim 17.

29. A method of inhibiting binding of a ligand to integrin _vβ₅ expressed on a cell in a subject comprising administering to the subject the amino acid sequence of claim 18.

30. A method of identifying a compound which binds integrin α_vβ₃ and or α_vβ₅ comprising selecting a compound to have a structure which mimics the binding domain of the amino acid sequence of claim 7.

31. A method of identifying a compound which binds integrin α_vβ₃ and/or _vβ₅ comprising selecting a compound to have a structure which mimics the binding domain of the amino acid sequence of claim 11.

32. A method of identifying a compound which binds integrin α_vβ₃ and/or _vβ₅ comprising selecting a compound to have a structure which mimics the binding domain of the amino acid sequence of claim 17.

33. A method of identifying a compound which binds integrin α_vβ₃ and or _vβ₅ comprising selecting a compound to have a structure which mimics the binding domain of the amino acid sequence of claim 18.

34. A method of screening for a compound that binds integrin αvβ3 comprising contacting vβ3 with the compound and with the amino acid sequence of claim 1, 7, 11, 12, 17 or 18 and determining whether the compound competitively inhibits the binding of the amino acid sequence to integrin vβ3.