CA2063055A1 - Peptides having thrombospondin-like activity and their therapeutic use - Google Patents

Abstract

Abstract of the Disclosure Compounds and compositions comprising fragments and synthetic analogs of human thrombospondin are provided together with methods for their use as thrombospondin-like agents.

Description

2 ~ ~ t3 Toch~ical ~ield The present invention relates generally to peptide fragments and synthetic analogs of thrombospondin (TSP) which retain thrombospondin-like activity. The peptides retain and mimic the bioactivity of TSP as a potent promoter or inhibitor of cell adhesion and attachment to different cell lines. The peptides find use as agents in inhibiting metastasis since TSP has previously been shown to mediate tumor cell metastasis presumably by mechanisms involving the cell adhesive domain of TSP. These peptides also find use in different biological and pharmaceutical applications such as:
(a) promoting and inhibiting cellular attachment to tissue culture flasks, ~b) promoting wound healing, angiogenesis, and implant acceptance, (c) use as agents for anti-platelet aggregation and use as diagnostic reagents in different therapeutic applications.

Bao~qround Thrombospondin (also known as thrombin sensitive protein or TSP) is a 450,000 molecular weight protein composed o~ three identical disulfide linked polypeptide chains (Lawler et al. J. Cell Biol (1986) 101:1059-71).
TSP is secreted by platelets in response to physiological activat~rs such as thrombin and collagen (~awler, J. Blood (1986) 67:112-123). TSP comprises 3% o~ the total platelet protein and 25% of the total platelet alpha granular protein (Tuszynski, G.P. et al. ~1985) J~ Biol. Chem. 260:12240-12245). Other cells also synthesize TSP including fibroblasts (Jaffe, E. A. et al., (1983) _atl. Acad. Sci. USA 80:999-1002), smooth muscle cells (Raugi, G. J. et al., (1982) J. Cell Biol.
95:351-354), and endothelial cells (McPhearson, J. et al.

2t~'9 J. Biol. C~em. 256:11330-11336). TSP has been found in certain tumor tissues, such as melanoma cells (Varani, J. et al., (1989) Clin. Expl. Metastais 7:319-329), -squamous lung carcinoma (Riser, B. L. et al., (1988) Exp.
Cell Res. 174:319~329) and breast carcinoma (Pratt, D. A.
et al., (1989) Eur. J. Cancer Clin. Oncol. 25:343-350).
In addition, the following tumor cells in culture have been shown to synthesize TSP: fibrosarcoma, rhabdomyosarcoma, glioblastoma, Wilm's tumor, neuroblastoma, teratocarcinoma, choriocarGinoma, melanoma, and lung carcinoma (Mosher, D. F., (1990) Annu.
Rav. Med. 41:85-97). A number of recent studies have shown that TSP plays a major role in cell-cell and cell substatum adhesion (Tuszynski, G. P. et al., (1987~
Seminars in ThrQmbosis Hemostasis (13:361~368, Mosher, D. ~., (1990) ~nnu. Rev. Med. 41:85-97). TSP promotes cell attachment, platelet aggregation, and lung tumor colony formation in a murine model of experimental metastasis (Tuszynski, G P. et alO, (1987) Science 236:1570-1573, Tuszynski, G. P. et al., (1988) Blood 72:109-115). The role of TSP in adhesion is further supported by the observation that the extracellular matrix of most tissues contains TSP.
TSP is composed of linear polypeptide domains that specifically interact with various macromolecules such as plasma and matrix components. For example/ T5P forms a complex with heparin tYabkowitz, R. et al. (1989) J. Biol. Ch m. 264:10888-1089~), fibrinogen (Tuszysnki, G. P. et al. (lg85) J. Biol. Chem. 260:12240-12245), collagen (Mumby, S. M. et al. ~1984) J. Cell Biol.
98:10888-10896, and plasminogen (Depoli. P. et al. (1989) Blood 73:976-902). The structure of TSP is conserved among various animal species as indicated by the fact - 4 - 2~3~$ J

that ~he antiobody aga~nst the human protein cross-reac~s with TSP from mouse, rat, pig, cow, sheep, dog, and turkey (Switalska, H. I. et al., J. Lab Clin. Med.
106:690-700).
Thrombospondin has been purified by a number or procedures including exclusion chromatoyraphy (Lawler et al., J. Biol. Chem. (1978) 253:8609-16), heparin affinity chromatography (Lawler ~t al., Thromb. Res. (1981 22:2~7-269)l fibrinogen affinity chromatography (Tuszynski et al., J. Biol. Chem. (1985) 260:12240-5), barium chloride precipitation (Alexander et al., Biochem.
J. (1984) 217:67-71) and anion exchange chromatography with HPLC (Clezarolin et al., J- ~b~gm3~g~. ~1984) ~96:2~9-56).
Ths complete amino acid sequence of TSP has been deduced from DNA clones prepared by various groups including Lawler et al., J. Cell Biol. (1986) 103:1635-48; Kobayashi et al., Biochamistry ~1986) 25:8418-25; Dixit et al., Proc. Ntl. Acad. Scl. (1986) 83:5449-53; and Hennessy et al., J. Cell Biol. ~1989) 108:729-36.
Cell adhesion is critical to the development and ~urvival of multicellular organisms. The process of cell adhesion is complex re~uiring numerous extracellular proteins such as ~ibronectin, vitronectin, collagen, laminin, and TSP and numerous families o~ cellular receptors such as the integrins and cellular adhesion molecules (CAMS). These molecules are involved in the adhesion of both normal and tumor cells and have been studied quite intensively in recent years.
The amino acid sequence, Arg-Gly-Asp (RGD), was established as a c~ll attachment domain in fibronectin (Pierschbacher, M.D. and Ruoslahti7 E., (1984) Nature _ 5 _ 2~

~London) 309:30-32). The same or related sequences have been discovered in many prot ins and serve as cell binding sites for such macromolecules as fi~rinogen (Ginsberg, M. D. et al., ~1985) J. Biol. Chem. 260:11891-11896). However, it appears that the adhesive function of laminin may not be based on the RGD sequence, but on a peptide segment of the B1 chain containing the amino acid sequence tyrosine-isoleucine-glycine-serine-arginine (YIGSR) (Sasaki, M. 1987, Proc. Natl. Acad. Sci 84:935-93~). Synthetic peptides containiny the RGD and YIGSR
sequence promote cell adhesion.
The therapeutic use of synthetic peptides based on the adhesive domains of fibronectin and laminin have recently been reported. Humphries et al. (2986) Science 233:467-470) were the first to demonstrate that co-injection of the pentapeptide GRGDS with B16-F10 murine melanoma cells dramatically inhibited the formation of lung colonies in C57BL/6 mice. Anoth~r synthetic peptide which was derived from laminin (YIGSR) also dramatically inhibited B16-F10 melanoma cell metastasis in C57B1/6 ~ice (Kleinman, H. K. et al., (1987) Science 238:1132-1133; Kleinman, H. K. et al., ~1990) Proc. Natl~ Acad. Sci. USA 87:2279-2283). The inhibitory activity of th~se peptides may be due to competition with endogenous laminin and fibronectin-dependent adhesion of tumor cells to the vascular bed of the target organ during the metastatic dissemination of the tumor cells.
Because metastasis is a step-by-step process involving the transfer of tumor cells ~rom one site to another through the lymphatic and blood circulation and p]atelet reduction in animals effectively blocked metastasis in animals (Gasic et al, (19683 Proc. Natl.

6 2 ~ ~ ~3~ i !, i Acad Sci USA 48:46-52), platelets have been thought to play a special role in the development of metastasis.
Since TSP comprises 25~ of the t.otal alpha granular platelet secreted-protein, TSP would be expe~ted to have a major role in the hemotagenous transfer o~ tumor cells to distant organs. Indeed, TSP has been shown to promote tumor cell metastasis in a murine model (Tus~ynski et al, (1987) 47:4130-4133). In addition, events which accompany platelet activation, such as: secretion of adhesive proteins, platelet aggregation, activation of proteolytic enzymes, and activation of the clotting cascade have all been shown to play a significant role in tumor cell metastasis ~Gasic, G. J., (1984) Cancer Metastasis Rev. 3.99-116).
Adhesive proteins which are part of the extracellular matrix control the movement, growth, and morphology of many cell types. Extracellular matrix proteins interact with tumor cell receptors and affect tumor cell adhesion to basement membrane collagen in different ways. For example, exposure of melanoma cells ln vitro to laminin resulted in increased capacity of tumor cells to adhere to the basement membrane and to produce lung tumor colonies (Barsky, S. H. et al., (1984) J. Clin. Inv. 74:843-848; Terranova, V. P. et al., (1984) science 226:982-985).
In view of the information described above, TSP may play an important role in many diverse and clinically important processes, such as; cell migration, wound healing, nerve regeneration, and tumor cell metastasis.
To better understand the pathophysiology of these processes at the molecular level, assignment of each of the biological activities of TSP to a specific subdomain or oligopeptide of TSP would provide valuable - 7 ~ 3~

information. Specifically, detailed knowledge of the structure of domains of the TSP and TSP receptors could be used to design TSP antagonist peptides which could block pathophysiological activities of TSP such as TSP-dependent tumor cell metastasis formation.
TSP contains three homologous peptide sequences designated type I, II, and III repeats (Lawler and Hynes, (1986) J. Cell Biol. 103:1635-1648). The three repeats consist o~ approximately 60 amino acids each containing six cysteine residues. Type I repeats exhibit homology with peptide segments found in a number of diverse proteins. We have identified a tetrapeptide sequence in TSP (VTCG) that i5 either totally conserved in other proteins or present with one or two conservative amino acid substitutions. The prevalence of the conserved sequences is indicated in Table I helow.

- 8 - 2~3~

TABLE I
Protein Sequence Re~erence TSP VTCG (SEQ ID No. 1) Lawler et al., (19~6), TSCG J. Cell Biol. 103:1635-48 Circumsporozoite VTCG Dame J. B. et al., (1984 ~ a~ 225:593~-5~9 Trap VTCG Robson K. J. H. et al., (19B8) Nature 335:79-82 Properdin VTCG &oundis, D. et al. (1988) Nature 335:82-85 Glycoprotein E VTCG McGoech D. J. et al, (1985) Herpes simplex I J. Mol. Biol. 181:1-13 Cytochrome C ETCG Lawson, J. E. et al., (1985) oxidase Curr. Genet. g:351 360 polypeptide II
Respiratory VTCX Blasco, F. et al. (1989) nitrate reductase Mol. Gen. Genet. 218:249-256 beta ~hain Bird spider 18S VSCG Hendriks 1. et al., (1989) ribosomal RNA Eur. J. Biochem. 177:15-20 Chicken alpha W CG Lemi~chka I. R. et al. (1981) tubulin J. Mol. Biol. 151:101-120 Zebrafish KTCG NjQ1Stad P. R. et a1., (1990) homeobox gene EMBO J. 9:515-524 E. coli gut VTCX Yamada M. et al., (1987) operon Biol. Chem. 262:5455-5463 E. C~li ~TPase VTCM Kanazawa H. et al., (19~1) BBRC: 103: 613 620 Rat liver VGCG Poncin J. E. et al., (1984) apolipoprotein Eu . J. Biochem. 140:493 A-I
Tryptophan VTCG Brosius, J. et al., ~1982) synthetase Gene 17:223-228 9 2~

Highlands J VTCL Ou J~ H. et al., (1982) virus J. Mol. ~iol. 156:719-730 Human c-myb VTCK 51amon D. J. et al., (1986) proto-oncogene Science 233:347-351 Antistasin RTCP Ginsberg V. et al. (1990)~HCP J~ Biol. Chem. 264:12138-Etpl00 CECG Tomley F. et al (198g) ATCG 5th International Coccisiosls RTCG Conference 469-573 EQCG
Human desmin VTCH Li Z. et al., (1989) Gene 78:243-254 Human related VPCG May F. E. B. et al., J. Virol mammary tumor 60:743-749 virus Human proto- RTCG Ouweland A. M. W. et al., oncogene c-fes/ EMBO J. 4:2897-2903 fps Platelet GPIIb VTCR Bray P. F. et al., (1987) J. Clin Invest. 80:1812-1817 _ The present invention provides thrombospondin fragments and analogs which mimic or inhibit the biological activity of intact thrombospondin which find use in a variety of biological, prophylactic or therapeutic areas.

~um~ry_~ the I~ventio~
It has now been found that a class of fragments or synthetic analogs from a specific domain of thrombospondin have a variety of uses. These peptides, by means of their adhesive activity, are capable of modifying and inhibiting tumor cell metastasis, cell - 10 - 2~3~

adhesion and platelet aggregation in mammals in vivo.
The peptides are also useful in wound healing, as diagnostic reagents and in other related areas. The peptides are capable o~ promoking cellular attachment and therefore can ~e used for preparing sur~aces for optimal cell culture, derivatization of various prosthetic materials, and to promote binding o~ surrounding tissues.
Medical devices can also be designed which make u~e o~
such substrates to attract cells to a surfacP in ViYo or even to promote the growth of a desired cell type on a particular surface prior to grafting. ~he TSP peptides and analogs of this invention have been shown to have thrombospondin-like activity.
The present invention is, there~ore, in one aspect directed to polypeptid~ compounds having thrombospondin-like activity which are identified by the formula:
Rl xl-x2-cYs-R2 wherein:
Rl is a protected or unprotected terminal amino group, including hydrogen, amino, acletyl or at least one amino acid residue or the desamino form thereof, preferably arginine;

Xl and X2 are the same or different neutral/non-polar/large/nGn-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residues, preferably selected ~rom the group consisting of valine, threonine, and serine;

R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide 2~$39.-~ i forms thereof, preferably selecte~ from the group consisting of lysine, glycine, and arginine.

Also provided in accordance with aspects of the invention are pharmaceutical compositions which contain the above-recited polypeptide compounds together with a pharmaceutically acceptablP liquid, gel or solid carrier.
Administration o~ therapeutically effective doses of these compositions can provide effective enhancement or inhibition of thrombospondin-like activity to animals, particularly vertebrates such as mammalian and avian hosts.

Bri~f Description of the Dr~yinq~
Figure 1 shows the ability of the peptides of the invention to inhibit adhesîon of B16Flo melanonma cells.
Figure 2 shows the ability of the peptides of the invention to inhibit adhesion of human lung caranoma cells.
Figure 3 shows the ability of the peptides of the invention to inhibit adhesion of bovine endothelial cells.
Figure 4 shows the ability of the peptides of the invention to inhibit adhesion of rabbit smooth muscle cells.
Figure 5 demonstrat2s that the peptides of the invention have antimetastatic activity ln vivo.

- 12 ~ 2 ~$3 _taile~ De~criPtion of th Tn~e~tion In accordance with the present invention, a class of fragments and analogs of khrombospondin is providsd which is capable of inhibiting or mimicing the activity o~
thrombospondin in mammals ln vivo.

A. _Definition3 "Thrombospondin-like activity" is defined herein as any activity that mimics the known biological activities of thrombospondin. These activities include cell-adhesion promoting activity, c~ll mitogenic activity, cell chemotactic activities, and hemostatic activities and any activities that derive from these activities such as tumor celll microbial, or parasite metastasis activity, platelet aggregating activity, fibrinolytic activity and immune modulation.
"Antimetastatic activity" is defined herein as the ability to prevent or greatly reduce the extent or size of tumor cell metastasis, or inhibit or cause regression of primary solid tumors.
"Wound healing activity" is defined herein as the ability to increase the rate at which wounds heal or to improve the results of the healing process (i.e., less scarring, good response to tactile stimulus, etc.;
"Angiogenesis activity" is defined herein as the ability to inhibit or enhance the formation of blood vessels or lymph vessels.
"Growth factor activity" is defined herein as the ability to inhibit or promote cell proliferation.
"Cell adhesion ackivity" is defined herein as the ability to promote or inhibit the attachment of cells, preferably mammalian cells, to a substrate.

~ ~3~ ~;

The sequence of amino acid residuss o~ the present polypeptide compounds, the core tetrapeptide, and preferred embod.iments thereof, are de~ined in terms of amino acids of certain characteristics of particular subclasses.
Amino acid residues can be generally subclassified into four major subclasses as follows:
Acidic, i.e~, the residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
Basic, i.e., the residue has a positive chargs due to association with H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptida is in aqueous medium.
Neutral/non-Polar, i.e., the residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide i5 in a~ueous medium.
Neutrallpolar, i.e., the residues are not charged at physiological pH and the residue i5 attracted by aqueous solution so as to seek the outer positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
It is understood, of course, that in a statistical collaction of individual residue molecules some molecules will be charged, and some notO To fit the definition of charged, a significant percentage (at least approximately - 14 - 2~ J

25%) of the indi~idual molecules are charged at physiological pH.
Amino acid residues can be further subclassified as cyclic or non-cyclic, a self-explanatory classification with respect to the side chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of khree carbon atoms or less. Small residues are, of course, always non-cyclic.
For the naturally occurring protein amino acids, subclassification according to the foregoing scheme is as follows:
Acidic: Aspartic acid and Glutamic acid;
Basic/non-cyclic~ Arginine and Lysine;
Basic/cyclic: Histidine;
Neutral/polar/small: Glycine, Serine and Cysteine;
Neutral/polar/large/non-cyclic: Threonine, Asparagine and Glutamine;
Neutral/polar/largeJcyclic: ~yrosine;
Neutral/non-polar/small~ ~lanine;
Neutral/non-polar/large/non-cyclic: Valine, Isoleucine, Leucin~ and Methionine~
Neutral/non-polar/large/cyclic: Phenyl~lanine and Tryptophan.
The protein amino acid proline, although within the classi~ication neutral/non-polar/large/cyclic, is not included as an alternative due to its known effects on the secondary conformation of peptide chains.
Certain commonly encountered non-natural amino acids, such as desamino Tyrosine (des Tyr), agmatine (Agm), n-formyl tryptophan (f-Trp), alpha-aminoisobutyric acid (Aib), and sarcosine (Sar), statine, ornithine 3 ~
(Orn), homolysine, homoserine, homoarginine, norleucine ~Nle), norvaline may also be incorporated into the compounds of the invention. Desamino tyrosine is incorporated at the N~terminus. Agmatine and statine are incorporated at the C-terminus. Based on the above definition, n-formyl Trp is neutral/non-polar/large/
cyclic, Sar is neutral/non-polar/small, Aib is neutral/non-polar/non-cyclic, Orn is basic/non-cyclic, homolysine is basic/non-cyclic, homoserine is neutral/polar/small, homoarginine is basic/non-cyclic, norleucine is neutral/non-polar/large/non-cyclic, and norvaline is neutral/non-polar/large/non-cyclic.
The nomenclature used to describe polypeptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the ri~ht of each aminc acid residue. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxy-terminal groups, although not specifically shown, will be understood to be in th~ form they would assume at physiologic pH valuesl unless otherwise specified. In th~ amino acid structure formulae, each residue is generally represented by a one-letter or three-letter designation, corresponding to the trivial name of the amino acid, in accordance with the following schedule:

.;J
Three-letter One-letter Amino Acid Symbol _ Symbol Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glycine Gly G
Histidine Hi~ H
Isoleucine Ile Leucine Leu L
Lysine Lys K
Methiona Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp Tyrosine Tyr Valine Val V

In the prasent application, the L form of any amino acid residue having an optical isomer is intended unless othexwise expressly indicated, e.g., by the sym~ol "[D-~]."
Compounds within the scope of the present invention can be obtained by modifying the disclosed formulae in numerous ways, while preserving the activity o~ the polypeptide compounds thu~ obtained. For example, while the amino acids of these compounds are normally in the natural L optical isomer form, one or more, usually two 2 ~ ~ 3 i j, or less and preferably one amino acid may be replaced with the optical isomer D form, or a D,~-racemic mixture can be provided in the mole~ules comprising the polypeptide compound.
Additionally, a disulfide linkage may be present or absent in the compounds of the invention, as long as activity i5 maintained. Rs one skilled in the art would recognize, branched or cyclic chains may be produced by the formation of a peptide bond with amino acid side groups that contain amino or carboxyl moieties. Amino acids containing such side groups include, for example, glutamic acid (carboxyl group), spartic acid (carboxyl group) and lysine (amide group3. Branched or cyclic chains may also be produced through the formatiGn of a covalent disulfide bond between amino acid residues having sulfur-containing side groups, such as cysteine.
As used herein, "protected" terminal amino group, refers to a terminal amino group coupled with any of various amino-terminal protecting groups traditionally employed in peptide synthesis. Examples of suitable groups include acyl protecting groups, for example, formyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromatic urethane protecting groups, for example, benzyloxycarbonyl; and aliphatic urethane protecting groups, ~or example, tert-butoxycarbonyl or adamantyloxycarbonylO Gross and Mienhofer, eds., The __des, vol. 3, pp. 3-88 ~Academic Press, New York, 1981), disclose num rous suitable terminal amino protecting groups.
As used herein, "protected" terminal carboxyl group, refers to a terminal carboxyl group coupled with any of various carboxy-terminal protecting groups. As will be readily apparent to one skilled in the art, suitable J~ 5 groups include tert-butyl, benzyl or other acceptable groups linked to the terminal caroxyl group through an ester or ether bond.
Amino acid re6idues contained within the compounds, and particularly at the carboxy- or amino- terminus, can also be modi~ied by methylation, amidation, acetylation or substitution with other chemical groups which can, for example, change the circulating half-life, resistance to proteases and solubility of the compounds without adversely effecting their activity.
In addition to the preceding definitions, the following abbreviations have been used throughout in describing the invention:
8CA bicinchoninic acid BSA bovine serum albumin t-Boc t-butyloxycarbonyl Bzl benzyl C degrees centigrade ~CM dichloromethane DIEA diisopropyl ethyl amine DMEM Dulbecco's minimum essential medium DME dimethyl formamide HF hydrogen fluoride HOBT l-hydroxybenzotriazole HPLC high performance liquid chromatography mBHA methylbenzhydrylamine ~g microgram ~l microliter ml milliliter mM millimolar nm nanometers 2 ~

NMP N-methylpyrrolidone % percent P~M phenylacetamidomethyl PBS phosphate buffered saline TFA trifluoroacetic acid B~ ~re~errea E~boaim0nt~
The polypeptide compounds of the invention all contain the core trip~ptide sequence:
Rl xl-x2-cYs-R2 wherein:
R1 is a protected or unprotected terminal amino group, including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof, preferably arginine;

X1 and X2 are the same or different nautral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polarjsm~ll amino acid residues, preferably selected from the group consisting of valine, threonine and serine;

R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl t or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, prefer~bly selected from the group consisting of lysine, glycine, and arginineO

Particularly preferred are those embodiments wherein the sequence is selected from the group consisting of:
VTCG (SEQ ID No. 1) RVTCG NH2 (SEQ ID No. 2) 2~3~

Compounds within the scope of the present invention san be synthesized chemically by means well known in the art such as, e.g., solid phase peptide synthesis. The synthesis is commenced from the carboxy terminal end of the peptide using an alpha-amino protected amino acid.
t-Butylocarbonyl (Boc) protective groups can be used for all amino groups even though other protective groups are suitable. See Stewart et alO ~ ~Solid-Phase Peptide Synthesis," W. H. Freeman Co., San Francisco (1969) and Merrifield, J. Am. Chem. Soc. 85: 2149-?154 (1963~, Vale et al., Science 213, 1394-1397 (1981), and Marke et al., J. Am. Chem. Sci. 103, 3178 (1981). Other preparative methods which may be employed include the process of Houghton Proc. Natl. ACAD Sci. 82:5132 (1981), or another preferable synthesis procedure particularly for small branched or cyclic chain peptides which would include conventional liquid phase processes. The liquid phase process, as well as other synthesis methods are described in "Principle of Peptide Synthesis" M. ~odansky Ed.
(Spring-Verlag 1984. These and other methods of peptide synthesis are also exemplified by U.S. Patents Nos.
3,~362,925, 3,842,067, 3,972,859 and 4,105,602, 4,683,291, 4,244,946, and 4,305,872.
Conveniently, compounds may be synthesized using manual techniques or automatically employing, for example, an Applied BioSystems 430A Peptide Synthesizer (Foster City, California) or a Biosearch SAM II automatic peptide synthesizer ~Biosearch, Inc., San Rafael, California), following the instructions provid~d in the in~truction manual supplied by the manufacturer.
Although a purity of greater than 95 percent for the synthesized p~ptide is preferred, lower purity may be acceptable. To obtain cyclic peptides, where for example ~ 21 ~ 2~ r i two cysteine amino acids are bonded or whers the residues contain a disulfide bridge which may be formed by oxidiziny of a dilute aqueous solution of the peptide with K3[Fe(CN)6]. Other means of cyclizing which are known in the art may also be utilized. The stabilized cyclized peptide of the present invention can also be prepared by forming a peptide bond between non-adjacentamino acid residue. A procedure for forming such peptide bond i5 provided in Schiller et al., Int. J.
Peptide Protein ResO tl985) 25:171.
It will be readily appreciated by those having ordinary skill in the art of peptide synthesis that the intermediates which are constructed in accordance with the present disclosure during the course of synthesizing the present compounds are themselves useful compounds and are thus within the scope of the invention.
~lternatively, selected compounds of the present invention can be produced hy expression of recombinant DNA constructs prepared in accordance with well-known methods. Such production can be desirable to provide large quantities or alternative embodiments of such compounds.

C. A~mini3tration Compounds of the present invention have thrombospondin-like activity in the intact animal.
Compounds of the present invention and compositions containing them which are shown to have the physiological effect of inhibiting or mimicing the effect of intact thrombospondin find use in numerous therapeutic and prophylactic applications, such as cancer therapy, wound healing, thrombotic or thrombolytic conditions, angiogenesis, or cell attachment.

- 22 ~ ~ , Thus the present invention also provides compositions containing an effective amount of compounds of the present invention, including the nontoxic addition salts, amides and esters thereof, which may, alone, serve to provide the above-recited therapeutic benefits. Such compositions can also be provided together with physiologically tolerable liquid, gel or solid diluents, adjuvants and excipients.
These compounds and compositions can be administered to animals for veterinary usel such as with domestic animals, and clinical use in humans in a manner similar to other th~rapeutic agents. In general, the dosage required for therapeutic efficacy will range from about 1 ~g to 300 mg/kg, more usually 10 ~g to 30 mgJkg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained.
Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The prepaxation may also be emul~ified. The active ingredient is often mixed with diluents or excipients which are physiologically tolerable and compatible with the active ingredient. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances such as wetting or emulsi~ying agents, stabilizing or pH buffering agents, and the like.

23 2 ~ ~b ~ ~ " ;,:i The compositions are conventionally administered parenterally, by injection, for example, either subcutaneously or intravenously. ~dditional formulations which are suitable for other modes of administration include suppositories, intranasal aerosols, and, in some cases, oral formulations. For suppositories, traditional binders and excipients may include, for example, polyalkylPIle glycols or triglycerides: such suppositories may be formed from mixtures containiny th~
active ingredient in the range of 0.5% to 10%, prefera~ly 1%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium sascharin, cellulose, magnesium carbonate, and the like.
These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain 10%-95% of active ingredient, preferably 25%-70~. These oral formulations include formulations designed to protect the peptide until it can be absorbed.
The peptide compounds may be formulated into the compositions as neutral or salt forms. Pharmaceutically acceptable non-toxic salts include the acid addition salts ~formed with the free amino groups) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, ox such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic ~ases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

- 24 ~ 3 ,,~,1"~

In addition to the compounds oE the present invention which display thrombospondin-like activity, compounds of the present invention can also be employed as intermediates in the synthesis of such useful compounds.
Tha compounds of the invention can be homopolymerized to themselves (i.~ peptide)n) or, heteropolymerized to one another ti.e., (peptide 1-peptide 2). The compounds can also be cyclized through disulfide or other means. The compounds can also be conjugated to hiocompatible polymeric ~ompounds, such as BIOPOL polymers (W. R. Grace & Co.-Conn.).
While not wishing to be bound by any theory, it is believed that thP compositions of the invention act as agonists or antagonists to native thrombospondin. These compounds are also believed to act as agonists or antagonists to circumsporozoite protein, thrombospondin related anonymous protein, antistasin, and properdin complement protein. Further, since the compounds of the invention are small in size (relative to intact thrombospondin) the properties which they exhibit are more likely to be specific in nature, as opposed to the actions of other generally adhesive compounds such as RGD
containing compounds (tha ~equence of which is found in over a hundred proteins1 and fibronectin. The side effects oP the peptide compounds of the invention are greatly reduced when compared with these broadly adhesive compounds.

D. U~e As stated previously, the compounds of the invention can be used in a variety of biolo~ical, prophylactic or therapeutic areas. It is con~emplated that these ~3 compounds are usPful in prevention or treatment of any disease state or conditions wherein thrombospondin-like activity plays a role. These disease states and conditions include, but are not limited to, metastasis, wound healing, thrombotic conditions, angiogenesis, and cell proliferation. Antibodies directed against the compounds of the invention are also useful as diagnostic reagents, therapeutics, or carriers of other compounds.
Tha compounds can also be used in biomedical devices.
Numerous 1n vitro and ln vivo assays can be used to demonstrate compounds having thrombospondin-like activity. These assays include, but are not limited to, cell adhesion assays, platelet aggregation assays and cell proliferation ~ssays.

NETA~;TA13Iæ
Metastasis is the spread of disease from one part o~
the body to another unrelated to it, as in the transfer of the cells of a malignant tumor by way of the bloodstream or lymphatic~. It is believed that metastasis is effected through a cascade mechanism which includes adhesion of tumor cells to endothelium, retraction of the endothelium, matrix degradation of the basement membrane and invasion of the tumor cells into the bloodstream. Intervention at any phase in this cascade could be beneficial to the treatment or prevention of metastatic cancersO
The native thrombospondin molecule has been shown to potentiate tumor cell metastasis ~Tuszynski et al., Cancer Research (1987) 47:4130-4133). The mechanisms by which the thrombospondin potentiation occurs are not presently well understood.

- 26 - 2 ~

Antimetastasis activity is characterized by the ability of the compounds to bind to melanoma cells ln vitro ~Tuszynski ek al., Anal. Bio. (1990) 184:189-91), and the ability to reduce the size and number of tumor colonies in vivo (Tuszynski et al. Cancer Research (1987 47:4130 4133)~
The compounds of this invention are useful as antimetastatic agents, particularly useful as anti pulmonary metastatic ayents. These compounds inhi~it the adhesion of metastatic tumor cells, particularly ~hose which are responsive to thrombospondin. The compounds also reduce tumor colony number as well as tumor colony size.
There are a number of mechanisms by which such antimetastatic activity can be occurring. The peptides can be cytotoxic, or inhibit cell proli~eration. As inhibitors of cell proliferation, the compounds can act to (1) inhibit mitogenesis, (2) inhibit angiogenesis, or (3) activate the complement pathway and the associated killer cells.
The compounds of the invention can also find use in biomedical devices. Since the compounds have the ability to promote the attachment of metastatic tumor cells, it is possible to coat a biomedical device with the compounds to effect the removal of circulating tumor cells from blood or lymph. The biomedical device is also useful to trap hepatomas.
~nother use o~ the compounds is as a carrier to target toxins, drugs, hormones or imaging agents to metastatic tumor c~lls for diagnostic or therapeutic purposes. These carriers would also bind to hepatomas.

2 ~

WOUND HEALIN~:
Wound healing is the closure of wounds and can be divided into ~our essential components: inflammation, angiogenesis, collagen deposition and epithelialization.
All four components play a role in the healing of wounds.
Wound healing activity is characterized by the ability of the compounds to show angiogenic activity, the ability of the compounds to stimulate collagen deposition and DNA synthesis in the 1n vivo sponge model or the ability of the compounds to improve wound healing or reduce healing time in an in vivo partial or full thickness wound model.

~N~I-PLATEL~ AGGR~GATION
Platelat aggregation is a normal and beneficial process to stop bleeding of damaged tissue. However, platelet aggregation can cause problems following cardiovascular treatment suc~ as angioplasty, thrombolytic therapy or vascular grafting. Platelets contain as much as 25% of the TSP protein in the total alpha granular platelet s creted-protein. Therefora, introduction of a peptide containing the pentapeptide sequence which is conserved in the TSP molecule and which binds to receptors on the surface of a platelet can prevent the p~atelet from aggregating and ~orming a clot.
A drug based on the pentapeptide is expected to be an adjunct to angiopla~ty and thrombolytic therapy for use with other clot-dissolv.ing agents which are currently in the market (e.g., tPA,streptokinan). Such an agent does not aggravate bleeding or have the risk of side eEfects common to synthetic anti platelet drugs.
Additionally, such a peptide can help to keep open small diameter vascular grafts tsuch as those used in heart by-- 28 ~

pass surgery). Similar appLications are envisioned for patients at risk for stroke.
Anti-platelet aggregation activity is characterized by a number of assays, including (1) inhibition of ADP
or thrombin-induced platelet aggregation in washed platelets; (2) inhibition of ADP-induced platelet aggregation in platelet-rich-plasma; and (3j inhibition of collagen induced platelet aggregation msasured in vivo.

1~7aIOG13NB~
Angiogenesis is the formation of blood and lymph vessels. Angiogenesis is essential during development, in wound healing and for the growth of solid tumor.
Angiogenesis is a complex process, requiring the sprouting and migration of endothelial cells, their proliferation and their differentiation into a tube like structure and the production of a basement membrane matrix around the vessel (Herbert et al. 1988, L. Cell, Biol. 106, 1365~1373). Angiogenesis is also essential to tumor development and growth and metastasis. Prevention of angiogenesis can inhibit solid tumor growth. Use of the compounds of this invention can inhibit one or more s~eps in the cascade process of angiogenesis and therefore such peptide may be useful clinically to inhibit metastasis. The compounds of this inv~ntion are useful in the modulation of angiogenesis, particularly in enhancing wound h aling, inhibiting or preventin~ tumor growth. The compounds of the invention are also useful in inhibiting or preventing diabetic retinopathy, neovascular glaucoma and rheumatoid arthritis. Standard angiogenesis assays are well known in the art. These assayas include, but are not limited to, proliferation - 29 - 2 ~ ~3~

and migration studies using various cell lines, collagenase inhibition and ln vlvo neovascularization on chicken chorioallantoic membranes (CAM assay).

ADHE~ION Y~D CIILL A'r~C~1!0311'r The peptides of the present invention can be used for preparing a surface for optimal cell culture, and for prosthetic materials to promote bonding with surrounding tissue. These peptides can be useful as a cell attachment protein to provide a substrate to which cells will attach by treating a hydrophobic surface such as untreated synthetic plastic resin and especially materials which are used for different membrane applications, e. g., nitrocellulose or polysulfone or comparable material with the peptide. The cell attachment properties of the peptides can also be used to couple polypeptides covalently to a solid support such as gels or synthetic resins or long chain polysaccharide.
This latter approach can be used for different affinity chromatography applications. Another important application of using such peptides are the use of the peptide in commercial cell attachment surfaces, wherein the particles are coated with gelatin, making it possible to grow the same adhere~t cells in a much smaller volume of media than would be possible in dishes. Medical devices can be designed for use with such peptides to attach cells to the surface in vivo, or even to promote the growth o~ a desired cell type on particular surfaces prior to grafting. An example of this is attachment of islet cells to a membrane or growth of endothelial cells on a prosthetic blood vessel or vascular graft. Such peptides can find uses in coating a patch graft or the like for aiding in wound healing.

- 30 ~ 3~

ANTIBODIES
Antibodies, both monoclonal and polyclonal, directed to peptide compounds of the present invention are useful in isolation and identi~ication of the subject protein from where the peptides are derived, and the present invention also pertains to such antibodies. To prepare antibodies, any one o~ a number of technigues which are known in the art can be employed~ In one such technique, polyclonal antibodies may he synthesized by injecting an animal (for example, a rabbit) with one or more compounds of the invention. After injection, the animal naturally produces antibodies to these compounds. When the antibody level rises to a sufficient level, antibody-containing blood, called antiserum, is then drawn from the animal, and the compound specific antibody is isolated from other antibodies in the antiserum by any one vf a number of separation techniques (for example, affinity chomatography). Monoclonal antibodies may be prepared using the technique of Kohler and Milstein, Nature 256, pp. 495-497 ~975).
Compounds of the present invention can also be used for perparing antisera for use in immunoassays employing labelled reagents, usually antibodies. Conveniently, the polypeptides can be conjugated to an antigen by means o~
dialdehydes, particularly from 4 to 6 carbon atoms and aliphatic, or carbodimide. These compounds and immunologic reagents may be labelled with a variety of labels such as chromophores, fluorophores such as, e.~, ~luorescein or rhodamine, radioisotopes such as l251, 35S,14C, or 3H, or magnetized particles, by means well known in the art.

- 3 1 ~ 3 ''~

These labelled compounds and reagents, or labelled reagents capable of recognizing and specifically binding to them, can find use as, e.g., diagnostic reagents.
Samples derived from biological specimens can be assayed for the presence or amount of substances havin~ a common antigenic determinant with compounds of the present invention.
Thrombospondin le~els are elevated in the sPrum of patients with metastatic breast and colon cancer (Tuszynski et al., Thrombosis Haemostas (1989) 62:4~8 and Smith et al., Proceedin~s American Association of Clinical Oncoloqy (19903 9:6). Antibodies against the peptides of the invention can be useful as reagents in diagnostic/prognostic assays for various types of cancer, including but not limited to, gastrointestinal tract (gastric, colonic, and rectal) carcinomas, breast carcinomas and hepatic carcinomas.
The polyclonal and monoclonal antibodies can find therapeutic use in a number of cancer therapies. First, the antibodies can be used to sequester thrombospondin.
This i8 useful since thrombospondin mediates tumor cell metastasis. Second, the antibodies can be used to block thrombospondin present on the tumor cell surface. Third, cytotoxic drugs, hormones, or imaging agents can be coupled to the antibodies for use in cancer therapy.
Fourth, a biomedical device can be coated with the anti~odies to remove excess thrombospondin from serum or the remove cells which bear thrombospondin on the ceIl surface.
The peptides of the invention can also be used to isolate thrombospondin cell surface receptors from extracts of cells or cell membranes. Standard procedures such as affinity chromatography can be employed. The - 32 - ~3~

thrombospondin cell surface receptors can be used to develop better thrombospondin analogs or to remove excess thrombospondi~ from serum.
The following examples are provided by way o~
illustration, rather than implying any limitation of the subject matter.

J~PJ.E~
The peptides of this invention can be synthesized by conventional methods of peptid~ synthesis. The preferred conventional methods use the procedures described in Int.
J. Pept. Proc. Res. 21, 57-63 ~1983). Also preferred is the solid phase synthesis of Merrified~ J. AmerO Chem.
Soc. 85, 2149-2154 (1963); Science 150, 178~185 (1965);
Ibid. 232, 3~1-347 ~1986). Solid phas~ synthesis is generally initiated from the C-terminal of the peptide by coupling a protected alpha amino acid to a suitable resin, e.g., phenylacetamidomethyl (PAM) polystyrene resin, or p-methylbenzhydrylamine mBHA) resin when synthesizing a peptide with a C-terminal carboxyamide.
During synthesisl suitable amino acid side-chain protecting groups are used as needed. Thus, aspartic acid is protected on the beta-carboxyl group as the benzyl ester and arginine is protected on the guanidino group by tosyl. After the desired peptide has been synthesized, the peptide is cleaved from the resin and protecting groups are removed by treatment with a reagent such as hydrogen fluoride (HF). The pepkide can then be purified by high performance liquid chromatography (HPLC) or other such methods of peptide purification.
BacXground information on the established procedures for solid phase peptide synthesis can be found in "Solid 2 ~ '.3 Phase Peptide Synthesis" by Stewart and Young, W. H. Freeman & Co., San Francisco, 196~.
In accordance with the above description, the following procedures were used for the chemical synthesis of novel synthetic peptides:

Example 1 Synthesis of the Pe~tide Sequence VTCG (SEQ ID No. 1 with C Terminal Amide -An appropriate resin 4-methylben~hydrylamine (MBHA) for C-terminal amide was sealed into polypropylene mesh packets (64~). All packets were placed into a common vessel with CH2C12 and vigorously shaken to wash and swell the resin. All subsequent steps involved vigorous 1~ shaking to ensure adequate solvent transfer. The N~a-butoxycarbonyl was then removed by acidolysis using 55% trifluoroacetic acid (TFA)/CH2C12 for 30 minutes leaving the ~-amino acid group in the TFA salt form. The packets were then washed with CH2C:L2 (2x), IPA (2x~, and CH2C12 (2x) to remove excess TFA and prepare for neutralization. ~he IFA alt was neutralized by washing the packets three times with ~% diisopropylethylamine in CH2C12 for 2 minutes each. This was followed by two washes with CH2C12 to remove excess base. Packets were then removed from the common vessel and added to their respective 0.2 M amino acid solutions which were prepared from computer generated information prior to neutralization. An equal volume of 0.2 M dipropyl~
carbodiimide was then added to activate the coupling reaction. After coupling at room temperature for 1 hour, the packets were washed with dimethyl- formamide and CH2C12 and returned to the common vessel. This process was repeated for each amino acid. Cleavage occurred by ~ 3 4 ~ 3 ~ ~ ~

reacting the peptide with 92.5% hydrogen fluoride/7.5%
anisole at -10C to 0C over 90 minutes. Anisole was used as a scavenger to react with carbocations produced as a result of the side chain protecting group removal.
This solution was then removed using a strong flow of M2 followed by the use of aspirator vacuum, while maintaining the temperature at 0C. Residual anisole was removed with 2 ethyl ether washes. The peptide was then extracted using 10% acetic acid.
Th~ purity of the crude peptide was checked by analytical RP-HPLC using a Beckman System Gold with a Vydac C-1~ column at a flow rate of lml/min. The solvent system used was Q.05~ aqueous TFA(A) and 0.05~ TFA in acetonitrile tB) with a gradient of 5-65% B in 30 minutes measuring the absorbance at 215 ~m). Purification was performed on Waters delta prep. 3,000 preparative HPLC
with a Waters prepO Pak Nodule Radial Compression C18 column (25 cm x 5 cm, 10-20 ~). The solvent syste~ was 0.05~ aqueous TFA (A) and 0.05% TF'A in acetontrile lB~.
Various linear gradients were used measuring the a~sorbance at 230 nm and collecting 40 ml fractionO The fractions were then analyzed on the Beckman analytical system. ~he desired fractions were pooled and lyophilized. The final dry product was analyzed one more time using analytical RP-HPLC.

Exam~le 2 Chemical Synthesis of the Peptide Sequence VTCG
(SEO ID No. 1) Acid An appropriate resin phenylacetamidomethyl (P~M) for C-terminal acid was sealed into polypropylene me~h packets (64~). All packets were placed into a common vessel with CH2Cl2 and vigorously shaken to wash and swell 2 ~ $ ~ / ?, , J

the resin. All subsequent steps involved vigorous shaking to ensure adequate solvent transfer. The N-~-butoxycarbonyl is then removed by acidolysis using 55%
trifluoroacetic acid (TFA)/CH2C12 for 30 minutes leaving the ~-amino acid group in the TFA salt for~. The packets were then washed with CH2C12 (2x), IPA (2x), and CH2Cl2 (2x) to remove excess TFA and prepare for neutralization.
The TFA salt was neutralized by washing the packets three times with 5% diisopropylethylamine in CH2C12 for 2 minutes each. This was followed by two washes with CH2Cl2 to remove excess base. Packets were then removed from the common vessel and added to their respective 0.2 M amino acid solutions which were prepared from computer generated information prior to neutralization.
An equal volume of 0.2 M diisopropylcarbodiimide was then added to ac~ivate the coupling reaction. After coupling at room temperature for 1 hour, the packets were washed with dimethylformamide and CH2C12 and returned to the common vessel~ This process was repeated for each amino acid. Cleavage occurred by reacting the peptide with 91~5% hydrogen fluoride/7.5~ anisole at -10C to 0C over 90 minutes. Anisole was used as a scavenger to react with carbocations produced as a result of the side chain protecting group removal. This solution was then removed using a strsng flow of N2 ~ollowed by the use of an aspirator vacuum, while maintaining the temperature at ooc. Residual anisole is removed with two ethyl ether washes. The peptide is then extracted using 10% acetic acid.
The purity o~ the crude peptide was ch~cked by analytical RP-HPLC using a Beckman System Gold wi~h a Vydac C-18 column at a flow rate of 1 ml/min. The solvent system used was 0.05% aqueous TFA(A) and 0.05%

- 36 - 2~

TFA in acetonitrile (B) with a gradient of 5 65~ B in 30 minutes measuring the absorbance at 215 nm.
Purification was parformed on Waters delta prep. 3,000 preparative HPLC with a Waters prep. Pak Nodule Radial Compression C18 column t25 cm x 5 cm, 10-20 ~). The solvent system was 0.05% aqueous TFA (A) and 0.05% TFA in acetonitrile (B). Various linear gradients were used measuring the absorbance of 230 nm and collecting ~0 ml fraction. The fractions were then analyzed on the Beckman analytical system. The desired fractions were pooled and lyophilized. The ~inal dry product was analyzed one more time using analytical RP-HPLC. Typical HPLC chromatogram for this peptide after purification are shown in Figure 2.
Example 3 Chemical SYnthesis of Additional Peptides Following the procedures outlined in Examples 1 and 2 and in Int. J. Pept. Proc. Res. 21, 57-65 (1983) with appropriate modi~ication, the following peptides were synthesized. All peptides of the invention were tested for endotoxins using standard LAL assay procedures and found to be endotoxin free:
RVTCG-NH2 (SEQ ID No . 2 ) Example 4 Adhesion of Various Cells to TSP and Peptides In this example a series of peptides were teæted to determine the abilities of the peptides to bind various cells as compared to thrombospondin. The cells used in this assay are B1~F1o melanoma cells, human lung carcinoma cells, bovine endothelial cells and rabbit smooth muscle cells. It is believed that thrombospondin acts in 37 ~ t, i metastasis through its adhesive properties. An assay was developed, generally in accordance with the disclosure of Tuszynski et al. (Anal. Bio. (1990) 184:18~-91) which evaluate~ the ability of cells to adhere to the thrombospondin fragments or analogs of the invention. In this assay, thrombospondin (purified by the method o~
Tuszynski et al., J. Biol. Chem. (1985) 260:12240-5) served as positive control, scrambled peptides VCTGSC, ANKHYF and TCVCGS served as negative contrsls.
Thrombospondin analoys of the invention were synthesized as described in Exampl~s 1-3. Two micrograms of peptide or control proteins were air dried overnight on the w~lls of a 96-well microtiter plate. Wells were then washed with HEPES-buffered saline and blocked ~or 1 hour with 1%
fatty acid free BSA in HEPES-buffered saline.
The various cells were grown and harvested during log phase of growth using standard procedures. The harvested cells were washed two times in serum-free Dulbecco's minimum essential medium (DMEM) (Flow Laboratories) and suspended in HEPES--bufferad saline, containing 5 mM glucos~ and 100 ~M MgCl2 at a final concentration of 2 x 105 cells/ml. Of the cell suspension 200,000 cells per well was added to each well of the microtiter dish containing the var:ious ligands and the dish incubated at 37C in a CO2 incubator for 30 minutes. Nonadherent cells were removed by aspiration and the wells ~ashed khree times with 200 ~l of PBS. The total cell-associated pxotein was determined by dissolving the attached cells directly in the microtiter wells with 200 ~1 o~ the Pierce BCA working solution ( i rce Chem. Co. Booklet No. 23225 (1987)). The plate was covered with an adhesive mylar sheet (Flow Labs) and incubated at 60C for 30 minutes. Plates were allowed to 2~$ ~
- 3~ -cool to room temperature, cover sheets were removed, and the absorbance of each well was determined at 562 nm with a microtiter plate reader (Biotek, Burlington, Vermont.) Absorbances were converted to number o~ adherent cells by means of an empirically determined conversion factox.
The results shown in Figures 1-4 indicate that the peptides of the invention display adhesive properties with respect to various cell lines.

Example 5 The Effect of Peptides on B16F1o Lung Tumor _ Cell Metastasi~
The ln vlvo model used to demonstrate the antimetastatic activity of the peptide compounds of the invention is described by Tuszynski et al. (Cancer ~es.
(1987) 47:4130-4133). Briefly, C57 black mice were intravenously injected with 1 x 105 B16F1o mouse melanoma cells in the presence of either control buffer (Hepes buffersd saline, pH 7O4)l or 1 mg of the designated peptide compound of the invention. Five or six animals were used for each compound. Peptides tested in the assay had no effect on cell viability as measured by Trypan blue dye exclusion. In addition, the peptides at 1 mg/ml did not effect cell growth after 24 hour~ o~
co~culture. After 14 days, the mice were sacrificed and the number of lung tumors counted.
The results shown in Figure 5 indicate the peptides of the invention have antimetastatic activity. Th2 bar graphs show the mean number of lung tumors observed in the treatment groups and the error bars represent the standard error of the mean.

39 ~3?U~CJl SEQVENCE LISTING

(1) GENERAL INFORMATION-(i) APPLICANT: Eyal, Jacob Hamilton, Bruce Ki~g Tusæynski, George Paul (ii) TITLE OF INVENTION: Peptides Having Thrombospondin-Like Activity and their Therapeutic Use (iii) NUMBER OF SEQUEMCES: 2 (iv) CORRESPONDENCR ADDRESS-(A) ADDRESSEE: W. R. Grace & Co.-Conn.

(B) STREET: 7379 Route 32 ~C) CITY: Columbia (D) STATE: Maryland (El COUNTRY: USA
(F) ZIP: 21044 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compati.ble (C) OPERATING SYSTEM: PC-DQSJMS-DOS
(D) SOFTWARE: Word Perfect 5.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
tc) CLA~SIFICATION: 530 (viii) ATTORNEY /AGENT INFORMATION:
(A) NAME: Appleby, Vanessa L.
(B) REGISTRATION NUMBER: 33223 (C) REFERENCE/DOCKET NUMBER: 01-7847 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (301) 531-4515 (2) INFORMATION FOR SEQ ID NO:1:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ~,Q~3~ ;t (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Val Thr Cys Gly (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPGLOGY: linear tii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Arg Val Thr Cys Gly.

AZAZ

Claims (31)

1. A method for promoting or inhibiting thrombospondin-like activity comprising administering an effective amount of a polypeptide compound comprising the formula:
R1-X1-X2-Cys-R2 wherein:
R1 is a protected or unprotected terminal amino group, including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof, preferably arginine;

X1 and X2 are the same or different neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residues, preferably selected from the group consisting of valine, threonine, and serine;

R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, preferably selected from the group consisting of lysine, glycine, and arginine.

2. The method of Claim 1 wherein the polypeptide compound is selected from the group consisting of:
VTCG (SEQ ID NO. 1) RVTCG-NH2 (SEQ ID NO. 2)

3. The method of Claim 1 which is useful for treating tumors.

4. The method of Claim 3 which inhibits tumor cell metastasis.

5. The method of Claim 3 which inhibits tumor growth.

6. The method of Claim 3 which reduces tumor size.

7. The method of Claim 3 which reduces tumor colony number.

8. The method of Claim 3 which inhibits adhesion of tumor cells.

9. The method of Claim 1 which promotes or inhibits cell adhesion.

10. The method of Claim 1 which promotes or inhibits chemotaxis.

11. The method of Claim 1 which promotes or inhibits hemostasis.

12. The method of Claim 1 which inhibits platelet aggregation.

13. The method of Claim 1 which is useful for promoting wound healing.

14. The method of Claim 13 which promotes or inhibits mitogenesis.

15. The method of Claim 13 which promotes or inhibits angiogenesis.

16. The method of Claim 1 which inhibits binding of a substrate to collagen.

17. The method of Claim 1 wherein the polypeptide compounds are homopolymerized or heteropolymerized, conjugated or cyclized.

18. The method of Claim 1 wherein said polypeptide compound is admixed with at least one pharmaceutically acceptable carrier prior to being administered.

19. A composition useful to promote or inhibit thrombospondin-like activity comprising a pharmaceutically acceptable carrier together with an effective amount of a polypeptide compound comprising the formula:
R1-X1-X2-Cys-R2 wherein:
R1 is a protected or unprotected terminal amino group, including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof, preferably arginine;

X1 and X2 are the same or different neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residues, preferably selected from the group consisting of valine, threonine, and serine;

R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, preferably selected from the group consisting of lysine, glycine, and arginine.

20. Antisera and antibodies which are capable of recognizing and specifically binding to an immunoreactive polypeptide comprising the formula:
R1-X1-X2-Cys-R2 wherein:
R1 is a protected or unprotected terminal amino group, including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof, preferably arginine;

X1 and X2 are the same or different neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residues, preferably selected from the group consisting of valine, threonine, and serine;

R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, preferably selected from the group consisting of lysine, glycine, and arginine.

21. The antisera and antibodies of Claim 20 which are useful as diagnostics.

22. The antisera and antibodies of Claim 21 which are useful to diagnose cancer.

23. The antisera and antibodies of Claim 20 which are useful as therapeutics.

24. The antisera and antibodies of Claim 23 which are useful to treat carcinoma metastasis.

25. The antisera and antibodies of Claim 20 which provide effective removal of TSP from circulating fluids.

26. The antisera and antibodies of Claim 20 which act as carriers.

27. The antisera and antibodies of Claim 26 which carry toxins, drugs, hormones, or imaging agents.

28. A method for facilitating the delivery of compounds to tumor cells for diagnostic or therapeutic purposes comprising the antisera and antibodies of Claim 20.

29. A method for facilitating the delivery of compounds to tumor cells for diagnostic or therapeutic purposes comprising a polypeptide compound comprising the formula:
R1-X1-X2-Cys-R2 wherein:
R1 is a protected or unprotected terminal amino group including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof, preferably arginine;

X1 and X2 are the same or different neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residues, preferably selected from the group consisting of valine, threonine, and serine;

R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, preferably selected from the group consisting of lysine, glycine, and arginine.

30. A biomedical device for promoting the attachment of tumor cells which incorporates a polypeptide compound comprising the formula:

R1-X1-X2-Cys-R2 wherein:
R1 is a protected or unprotected terminal amino group, including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof, preferably arginine;

X1 and X2 are the same or different neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residues, preferably selected from the group consisting of valine, threonine, and serine;

R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, preferably selected from the group consisting of lysine, glycine, and arginine.

31. A biomedical device for removal of thrombospondin which incorporates the antisera and antibodies of Claim 20.