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CA2134655A1 - Modulation of thrombospondin-cd36 interactions - Google Patents

Modulation of thrombospondin-cd36 interactions

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CA2134655A1
CA2134655A1 CA002134655A CA2134655A CA2134655A1 CA 2134655 A1 CA2134655 A1 CA 2134655A1 CA 002134655 A CA002134655 A CA 002134655A CA 2134655 A CA2134655 A CA 2134655A CA 2134655 A1 CA2134655 A1 CA 2134655A1
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polypeptide
thrombospondin
tsp
ligand
binding
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Russell J. Howard
Lawrence L-K. Leung
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Leland Stanford Junior University
Merck Sharp and Dohme LLC
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    • C07ORGANIC CHEMISTRY
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    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

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Abstract

Ligands are provided which specifically bind to human thrombospondin. Methods are also provided which employ the ligands to modulate the interactions of thrombospondin with platelets and cells bearing receptors for thrombospondin.

Description

wo s3t22340 2 1 3 ~ 6 5 ~i Pcr/uss3/03748 MODULATION QF THROMBQSPONDIN CD~6 INTERACIIONS
This application relates to thrombospondin and the receptor therefor, known as CD36, GPIIIb or GPIV, and to ligands 5 and methods which can be used to either augment or inhibit interactions between thrombospondin and the receptor. This application also relates to platelets and various cells bearing thrombospondin receptors.
BACKGROUN~ OF ~ ~V~TION
The specific binding of adhesive proteins to cell surface receptors or ~o ex~acellular matIices is an essential element of multiple cell adhesion phenomena such as tissue development, the immune response, vascular hemostasis and inflammation. A number of adhesive proteins, which may be 1~ pleiotropic and show significant functional redundancy, are involved in such processes.
One important mechanism in the generation of adhesion specificity is the induction of a high-affinity binding - state in the cell surface receptor following cell stimulation~ Thebest characteri~ed examples are induction of fibrinogen-binding -~
- function in the IIb~3 (GPIIb-IIIa) integrin on platelets [Phillips e~ al., Cel} 65:359 (1991~ u---et~al., Cell 65:40~ (1991)~ and the capacity for ICAM-1 binding by LFA-1 on T cells [Dustin et al., Natl~re 341:619 (1989)].
There is now~ evidence that cell adhesion can involve complex conformational- changes in the cell surface receptor induced by the ligand.--EQ~ example, binding of the recognition sequences Arg-Gly-As~- and the gamma carboxyl terminal dodecapeptide of fibnnogen to the IIb,B3 integrin induces 3û conformational changes in this receptor, generating a high-affinity fibnnogen-binding state.

W093/22340 21346~S PCl'/US93/03748 Thrombospondin (TSP) is a multi-domain adhesive macromolecule that interacts with a number of proteins, including fibrinogen, ~lbronectin and collagen [Leung et al., J. Clin. InYest. 70:542 (1982); Lahav et al., Cell31:253 (1982)].
5 TSP is a major platelet a granule protein and is released and expressed on activated platelet surfaces ~Phillips et al., J. Bioi.
Chem.255:11629 (1980)~. By interacting with fibrinogen, TSP
serves to reinforce platelet-platelet cohesion and to stabilize platelet aggregates [Leung, J. Clin. Invest. 74:1764 (1984)].
TSP is also synthesized by a variety of cell types and is incorporated into cell mal;rices, where it may modulate cell adhesion, mobility and growth. TSP also supports adhesion of Plasmodium falciparum-infected erytbrocytes and has been implicated in the pathogenesis of cerebral malaria [Roberts et al., 1~ Nature318:64 (1985); Howard et al.,Blood 74:2603 (1989)]-CD36 lalso known as glycoprotein IIIb (GPIIIb) or glycoprotein IV (GPIV)] is an integral membrane protein present on platelets, endothelial cells, monocytes, erythroid precursors, epithelial cells: and some tumor lines ~Knowles e~ al., J. I~ununol.
20 132:2170 (1984); Asch et al., J. Clin. Invest. 79:1054 (1987);
Kieffcr et al., Biochem. J. 262:835 (1989); Greenwalt et al., Biochem~stry 29:7054 (lg90); Catimel et al., Blood 77:2649 (1991)]. CD36 is one of the cell surface receptors for TSP, and the CD36-TSP interaction plays a role in media~ng platelet--~ 2~ -- pi:atolet and platelet-monocyte cell adhesion [Asch et al., supra;
McGregor et al., J. Biol. Chem. 264:501 (1989); Silverstein et al., J.
Clin. /n~est. 84:546 (19~9)], as well as cell attachment to matrix.
- - In such interactions, TSP acts as a bridge.
A substance that inhibits new blood vessel formation .
3~- :(an angiogenesis inhibitor) has been found to be a fragment or an isoform of TSP ~Good et al., Proc. Natl. Acad. Sci. USA 87:6624 (1990)]. TSP, or a particular isoform of it, may therefore play a wo 93/22340 ~ 1 3 ~ 5 ~ Pcr/uss3/'~3748 role in reguiating cell growth by this mechanism. The receptor site for this TSP isofonn has not been de~med but may be CD36.
The isolation and charac~erization of CD36 from platelets has been described by McGregor et al., supra; Tandon 5 et al. [J. Biol. Chem. 264:7570 (1989)] and Tsuji et al. [J. Biochem.
100:1077 (1986)]. ~::
In addition to serving as a receptor for TSP, CD36 can also function as a receptor for the adhesion of Plasmodil~m falciparum-infected erythrocytes [Barnwell et al., J. Immunol.
10 135 :3494 (1985); Ockenhouse et al., Science 243 :1469 (1989)].
, This interaction is mediated by a CD36 recognition protein which has recently been identified on parasitized cells. CD36 also reacts with OKM5, an anti-CD36 monoclonal antibody which has , been reported to inhibit the cell adhesive functions of CD36, 1 ~"
1~ including the in ~itro binding of Plasmod~um falciparum-infected erythrocytes to monocytes, endothelial cells and C32 melanoma cdls (Barnwell et al., supra). suggesting that the OKM5 epitope on -CD36 is functionally important. , i ' Rccently, the sequence Ser-Val-Thr-Cys-Gly has bcen 20 identified as a cell adhesi~rc motif in TSP having homology to the - ' maralia circumsporozoite protein [Prater et al., J. Cell Biol.
112:1031 (1991); Rich e~ al., Science 249:1574 (1990)].
CD36 also binds, a- serum protein called platelet agglutina~ng protein p~7, which is found in the sera of 25 thrombotic thrombocytopenic purpura patients. Platelet agglutination protein p3~ causes platelet agglutination through binding to membrane CD3~ ~[Lian et al., Thrombosis and Haemostasis 65:102 (199l)].
. _ _ Results of-,r,e,cent~ studies calTied out by Tr~zzini et al.
3~ [Immunology 71:29 (1990)] suggest that CD36 may even act as a signaling molecule i~ human monocytes.

WO 93/22340 PCI/US93/0374~
21~655 4 Because interac~ions between TSP and platelets or cells bearing receptors for TSP are involved in many important biological processes and disease states, there is a need for matenals and methods to modulate such in~eractions.
5 SUMMARYOF I~IN~T~
The present invention fulfills this need by providing ligands which selectively bind to a region of thrombospondin that specifically binds to a polypeptide having an amino acid sequence de~med by SEQ ID NO: l.
This invention further provides ligands which selectively bind to a region of thrombospondin, the presence of which region is induced by the binding to ~hrombospondin of a polypepdde having an an~ino ac;d se~uenee defined by SEQ II) NO~
This invention still further provides methods for augmenting thrombospondin-mediated effects comprising contacting- thrombospondin in the presence of platelets or cells bearing receptors for thrombospondin with an effective amount of a ligand which selectively binds to a region of thrombospondin -20- that specifically binds to a polypeptide having an amino acid ~ - sequence defined by SEQ ID NO~
This invention still further provides metbods for ~~- inhib~'ting thrombospondin-mediated effects comprising contacting thrombospondin in the presence of platelets or cells 25 bearing receptors for thrombospondin with an effective amount - -of- a ligand which selectively binds to a region of thrombospondin, the presence of which region is induced by the - _- bi~di~g to thrombospondin of a polypeptide having an amino acid sequence defined by SEQ ID NO: l.
2 1 3 4 6 5 ~ PCI`/VS93/03748 - Pharmaceutical compositions comprising one or more of the ligands and a pharmaceutically acceptable carner are also provided by this inven~ion.
BREF DESCRIPTION OF ~ FIGIJRES
This invention can be more readily understood by reference to the accompanying Figures, in which:
Fig. 1 is a schematic representation of a proposed model of CD36-thrombospondill interaction.
Fig. 2 is a g~aphical representation of the effects of 1 0 polypeptides P93-1 10 and P139-1~5 on the binding of CD36 to ~;
thrombospondin.
Fig. 3 shows platelet aggregation ~acings for human platelets tr~ated with ~rarious polypeptides plus ADP (A) or csll~gen (B) and (C).
Fig. 4 shows platelet aggregation ~acings far human platelets treated with collagen plus control buffer, antibodies from hybridoma subclone 7AIh, or various amounts of antibodies from subclone 7AIe.
Fig. 5 is a graphica~ representation of the binding of 12~I-P93-110 to thrombospondin, alone and in the presence of va~ious polypepddes.
DE~CRIPI IO~ OF T~ INV~NTION
All references cited herein are hereby incorporated in their entirety by reference. -As used herein, the:terrr~nligand" means a molecule which speci~lcally binds to human-TSP. Preferably, the ligands of ~e invention are antibodies or linear polypeptides, although cyclic peptides ~nd non-peptide organic compounds that bind to : : :

wo 93/22340 2 1 3 ~ ~ ~ 5 6 Pcr/uss3/03748 TSP and exhibit the activiaes described below are also contemplated by this invention.
The term "region of thrombospondin" is defined herein to mean a localized se~uenee of amino acids on the 5 surfaee of human TSP to which a ligand can bind.
The terms "poIypeptide" and "peptide" are used interchangeably herein and are intended to mean the same thing.
The term "specifically binds" is defimed in the context 10 of this invention to mean the binding of a ligand to human TSP
which is mediated by noncovalent short-range interactions, including but ~ot limited to hydrophobic7 ionic~ hydrogen bonding and van der Waals interactions. Such interactions are determined by spec;fic amino acid sequences and/or organic 15 compound functional groups.
As used herein, the term "selectively binds to a region of thrombospondin" means that the binding of a ligand to human TSP is both specific and localized to a distinct region of the TSP. Tbis definidon specifically e~cludes molecules such as 20 CD36, ~he binding of which is specific but not selec8ve because they~~ind to--~TSP at more than one region.
Th_ term "receptor for thrombospondin" is defined to mean~an am~no acid sequence on a platelet or cell which specifically bi~ds to human TSP. One such receptor is known as 25 CD36, GPmb or GPIV.
.
Ligands are provided by this invention which can either augment or inhibit TSP-mediated ef~ects. .
The ligands of this invention which augment TSP-mediated effects are exemplified by a polypeptide 30 designated Pl39-155 having an amino acid sequence defined by SEQ ID NO: l. This polypeptide represents an epitope on CD36 wo 93/2234~ 3 ~ fi .S 5 PCr/US93/03748 which is specifically recognized by monoclonal antibody OKM5.
Since OKM5 is known to inhibit TSP-CD36 interac~ons, it would be expected that the polypeptide, by binding to TSP, would do so as well. Yet, surprisingly, the binding of P139-15~ to TSP has 5 the opposite effect -- it actually augments the interaction between TSP and its receptor.
The inhibitory ligands of the invention are exsmplified by a polypeptide designated P93-l l0 having an arnino acid sequence defined by SEQ ID NO: 2. This polypeptide 10 binds to a site on TSP which is distinct from the site to which P139-155 binds. Surprisingly, the binding of Pl39-lS5 to TSP
induces the appearance of the site to which P93-ll0 binds, thereby greatly increasing the binding of the latter polypeptide.
A proposed model of the interactions between TSP j 15 and CD36 based upon the observed activities of the exemplary polypepdde ligands of the invention is shown in Fig. 1, although whether this model is correct or not is not essential to this invention.
Panel A of Fig. l shows that CD36 is believed to 20 contain two regions which can bind to TSP. The binding of one of these regions (represented by polypeptide Pl 39~ ) oecurs first, with later binding to TSP by the second region of CD36 (represented by polypeptide P93-110).
Prior binding of a ligand to TSP -at ttle site to which 25 polypeptide Pl39-155 binds causes induction of the other site on TSP and the binding of CD36 to the induced site (Panel B).
Because the affinity for binding at the-~induced site is believed to be greater than at the other site, the- nct effect is augmentation of the TSP-CD36 interaction.
Binding of a ligand to the site at which polypeptide P93-ll0 binds, however, prevents- CD36 from binding at that site (Panel C). Because the af~mity of binding of CD36 to the other WO 93~22340 2 1 3 ~ 6 5 ~ PCI`/US93/03748 TSP sitè is believed to be much lower, the net effect is inhibition of the interaction between TSP and CD36.
Although polypeptide ligands having amino acid sequences defined by SEQ ID NOs:l and 2 aro preferred for use in modulating the interactions between TSP and its receptors, those skilled in the art will appreciate that polypeptides for use in thi$ invention might also contain more or fewer amino acid residues based on the known sequence of CD36, as long as function is not substantially impaired. They may also contain residues not normally present in the soquence of CD36, as shown in the Example below.
As explained above, the ligands of the invention encompass more than polypeptides and include other molecules such as andbodies as ~well. For example, a monoclonal antibody ; 15 describod~below exhibited the same effects on TSP-CD36 ¦
interactions as did~polypeptide P93-llO. This antibody is characterized by about 5 fold greater binding to human TSP to whic:h has been bound a polypeptide having an amino acid ¦
sequence defined by SEQ ID NO: 1, compared to TSP to which ~-; 20 such a polypeptide has not been bound. This binding differential provides a convenient basis for screening hybridomas producing other~ ~antibodies having a similar specificity for binding TSP.
The ligands of this invendon may be useful for modulating TSP~36`interactions in any process in which such - ~ 25 interactions are-known or can be shown to occur.
For example, as noted aboYe, Plasmodium falciparum-infected erythrocy~e adhesion to microvascular cndothelial cells is believed to be mediated by infected cell .
recognitiorL~ CD36. There is also evidence that CD36 on tumor - 30 cells media-tes--adhesion to TSP lAsch e~ al., J. Biol. Chem.
266:1740 (1991)]. The ligands of the invendon which inhibit `
TSP-CD36 inte~actions may be used to block or revcrse such adhesion.
.

WO 93/22340 PCI'/US93/03748 21~655 There is also evidence that TSP-CD36 interactions play a role in stabilizing platelet aggregates ~Asch et al., ~. Clin.
Invest. 79:1054 (1987); McGregor et al., ~. B~ol. Chem. 264:501 (1989)~. The ligands of the invention which inhibit TSP-CD36 5 interactions may be thus also be useful for reducing or prevendng platelet aggregation. Such use of these ligands provides a novel approach to the design of and-platelet and anti-thrombotic agents.
Current efforts are mainly focused on the disruption 10 of the platelet aIIb,B3 (GPIIb-IIIa) interaction~ with fibrinogen, using monoclonal andbodies or polypeptides based on the integrin sequence and Arg-Gly-Asp-containing analogs [Yasuda et al., J. Clin. lnvest. 81:1284 ~1988); Shebuski et al., J. Biol. Chem.
264:21550 (1989); Scarborough et al., J. Biol. Chem. 266:9359 (199lj]. Although such and-platelet agents are functionally ¦
quite potent, they also have the potential to produce undesired 11 ' bleeding, as evidenced by a marked prolongation of the bleeding time (Yasuda et aL, supra; Shebuski et al., supra).
By interfering with the secondary phase of platelet 2:0 aggregation while keeping the primary phase functionally intact, use of the ligands which inhibit TSP-CD36 interactions may provide a new therapeutic approach- whic}i -minimizes the bleeding diathesis.
Finally, the use of the polypeptide ligands of this 25 invention which augment TSP-CD36~interactions may be useful for enhancing platelet aggregation in situations where such ~' aggregation is desirable. Such a situation arise in the treatment of pa~ients with funcdonal disorders or immune-mediated thrombocytopenia who cannot tolerate conventional platelet 30 transfusions.
Currently, platelet transfusions are given to bleeding patients who lack platelets, and desmopressin (DDAVP) is given to those who have functionally defective platelets. In the case of WO 93/22340 2 1 3 4 6 ~ 5 PCI'/US93/03748 the former treatment, however, some patients are refractory due to an existing autoimmune disorder or will become refractory due to alloimmunization. In the case of DDAVP treatment, it is known that the effect is temporary and cannot be used on a 5 long-term basis.
The polypeptide ligands which augment TSP-CD36 interactions in the case of platelets do not sct by themselves but instead enhance ADP- and collagen-induced platelet aggregation in platelet-rich plasma.
The polypeptide ligands of the invention are synthesized by a suitabie method such as by exclusive solid phase synthesis, pardal solid phase methods, fragment condensation or classical solution synthesis. The polypepddes ~- arc preferably prepared by ~solid phase peptide synthesis as described by Merrifield, J. Am. Chem. Soc. 85:2149 (1963).
- The synthesis is carried out with amino acids that are protected at thc alpha-arnino terminus. Trifunctional amino ' acids with labile sidc-chains are also protected with suitable groups t o prcvent undesired chemical react~ons from occurring during the asscmbly of the polypeptides. The a1pba-amino protecdng group is selectively removed to allow subse~uent~ reaction to- t~ke place at the amino-terminus. The conditions for the removal of the alpha-amino protecting group do not remove~ the= side-chain protecting groups.
The alpha-amino protecting groups are those ~known to be useful in the art of stepwise polypeptide synthesis. Included are acyl type protecting groups (e.g., formyl, trifluoroacetyl, acètyl), aromatic urethane type ;
protecdng groups lGg.~ ~ benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyI and 9-fluorenylmethyloxycarbonyl (Fmoc)], aliphadc urethane protecting groups (e.g., I
t-butyloxycarbonyl (Boc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protecting groups (e.g., W093/22340 ~ 4 5~5 5 1 1 pcr/us93~o3748 benzyl, triphenylmethyl). The prefelTed protecting group is Boc. The side-chain protecting groups for Tyr include tetrahydropyranyl, tert.-butyl, trityl, benzyl, Cbz, 4-Br-Cbz and 2,6-dichlorobenzyl. The preferred side-chain protecting 5 group for Tyr is 2,6-dichlorobenzyl. The side-chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl and cyclohexyl. The preferred side-chain protecting group for Asp is cyclohexyl. The side-chain protecdng groups for Thr and Ser include acetyl, benzoyl, tntyl, 10 tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl and Cbz. The pre~e~Ted protecting group for Thr and Ser is benzyl. The side-chain protecting groups for Arg include nitro, Tos, Cbz, adamantyloxycarbonyl and Boc. The preferred protecting group for Arg is Tos. The side-chain amino group of Lys may 15 be protected with Cbz, 2-Cl-Cbz, Tos or Boc. The 2-Cl-Cbz group is the preferred protecting group for Lys.
The side-chain protecting groups selected must remain intact during coupling and not be removed during the deprotection of the amino-terminus protecting group or 20 during coupling conditions. The side-chain protecting groups must also be removable upon the completion of synthesis, - using reaction conditions that will not alter the finished polypeptide.
Solid phase synthesis is usually carried out from 25 the carboxy-terminus by coupling the alpha-amino protected (side-chain protected) amino acid to a suitable solid support.
An ester linkage is formed when the attachment is made to a - chloromethyl or hydroxymethyl resin, and the resull:ing polypeptide will have a free carboxyl group~ at the C-terminus.
30 Alternatively, when a benzhydrylamine or--p-methylbenz-hydrylamine resin is used, an amide bond=is~- foTrned and the resulting polypeptide will have a carboxamide group at the C-terminus. These resins are commercially available, and their preparation has described by Stewart et al., "Solid Phase wo 93/22340 2 i 3 ~ 6 5 S PCr/US93/03748 Peptide Synthesis" (2nd' Edition), Pierce Chemical Co., Roclcford, IL., 1984.
The C-terminal amino acid, protected at the side-chain if necessary and at the alpha-amino group, is coupled to the benzhydrylamine resin using various activating agents including dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide and carbonyldiimidazole. Following the attachment to the resin support, the alpha-amino protecting group is removed using trifluoroacetic acid (TFA) or HCl in dioxane at a temperature between 0 and 25C.
Dimethylsulfide is added to the TFA after the introduction of methionine (Met) to suppress possible S-alkylation. After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled stepwise in the required 1~ order to obtain the desired sequence.
Various activating agents can be used for the coupling reactions including DCC, N,N'-diisopropyl-carbodiimide, benzotriazol-1-yl-oxy-tris-(dirnethylamino)- i;
pho~phonium hexafluorophosphate (BOP) and DCC-hydroxybenzo~iazole (HOBt). Each protected amino acid is ' used in excess (~2.0 equivalents), and the couplings are usually carried- out in~-N-methylpyrrolidone (NMP) or in DMF, CH2C12 or mixtures thereof. The extent of completion of the '--coupling reaction is monitored at each stage, e.g., by the ';
ninhydrin reac'tion' as- described by Kaiser et al.,Anal.
Biochem., 34:595 (1970). In cases where incomplete coupling is found, the coupling reaction is repeated. The coupling '`
reactions can be -performed automatically with commercially available instruments. I'' A~er-~t~ie-~'entire assembly of the des*ed polypepdde, 'the polypeptide-resin is cleaved with a reagent i -such as liquid E~ for 1-2 hours at 0C, which cleaves the polypeptide from the resin and removes all side-chain WO 93/Z2340 PCI'/US93~03748 4fi~
protecting groups. A scavenger such as anisole is usually used with the liquid HF to prevent cations formed during the cleavage fom alkylating the amino acid residues present in the polypeptide. The polypeptide-resin may be deprotected with TFA/dithioethane prior to cleavage if desired.
Side-chain to side-chain cyclization on the solid support requires the use of an orthogonal protection scheme which enables selective cleavage of the side-chain functions of acidic amino acids (e.g., Asp) and the basic amino acids (e.g, Lys). The 9-fluorenylmethyl (Fm) protectipg group for the side-chain of Asp and the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group for the side-chain of Lys can be used for this purpose. In these cases, the side-chain protecting groups of the Boc-protected polypeptide-resin are selectively rcmoved with piperidine in DMF. Cyclizadon is achieved on the solid support using various activating agents including DCC, DCC/HOBt or BOP. The ~; reaction is carried out on the cyclized polypeptide-resin as described above.
Recombinant DNA methodology can also be used to prepare the polypeptides. The known genetic code, tailored if _ desired for more efficient expression in a givea host organism, can be used to synthesize oligonucleotides en~odin-g-the - desired amino acid sequences. The phosphoramidite solid support method of Matteucci e~ al. [J. Am. Chem! Soc. _ - 2~ 103:3185 (1981)~, the method of Yoo et al. [J. Biol. .Chem.
764:17078 (1989)], or other well known methods can be used for such synthesis. The resulting oligonucleotides can be inserted into an appropriate vector and expressed- in a compatible host organism.
The polypeptides of the inventio~~can~~~e purified using HPLC, gel filtration, ion exchange and partition chromatography, countercurrent distribution or other known methods.

WO 93/22340 PCl'tUS93/03748 The an~ibody ligands of the invention which inhibit TSP-CD36 interactions can be prepared against TSP
using methods described below. Although polyclonal antiserum could in principle be used, monoclonal antibodies ;--are preferred.
Antibodies can be produced by immunizing a host animal such as a rabbit, rat, goatt sheep, mouse, etc. with TSP.
Preferably, one or more booster injections are given after the initial injection, to increase the antibody titer. Blood is then 10 drawn from the animal and serum is prepared and screened by standard methods such as enzyme-linked immunosorbent assay (ELISA) using TSP as the antigen.
The immunogenicity of the TSP used for i -immunization can be increased by combination with an adjuvant and/or by conversion to a larger form prior to immunization. ~ -Suitable adjuvants for the vaccination of animals include but are , -not limited to Adju~rant 65 (containing peanut oil, mannide monooleate and aluminum monostearate); Freund's complete or ~;
incomplete adjuvant; mineral gels such as aluminum hydroxide, 20 aluminum phosphate and alum; surfactants such as hexatecylamine, octadecylamine, lysolecithin, dimethyldioctad-ecyl-ammonium bromide, N,N-dioctadecyl-N',N'-bis(~-hydroxymethyl) propanediamine, ~
methoxyhexadecyl~lycerQl and pluronic polyols; polyanions such 25 as py~an, dex~an~ sulfàte,- poly IC, polyacrylic acid and carbopol;
pepddes such- as- muramyl dipeptidè, dimethylglycine and tuftsin; and oil emulsions. The polypeptides could also be administered following incorporation into liposomes or other microcarriers. - --., Monocional antibodies can be prepared using ;
standard methods, e.g., as described by Kohler e~ al. [Nature 256:495 (1975); Eur. J. Immunol. 6:511 (1976)~. Essentially, an animal is immunized ~ as described above to produce .

WO 93/22340 2 1 3 ~ ~ 5 ~ PCr/US93/0374X

antibody-secreting somatic cells. These cells are then removed from the immunized animal for fusion to myeloma cells.
Somatic cells with the potential to produce antibodies, -particularly B cells, are suitable for fusion with a myeloma cell 5 line. These somatic cells may be derived from the lymph nodes, spleens and peripheral blood of primed animals. In the exemplary embodiment of this invention rat spleen cells are used, in part because these cells produce a relatively high percentage of stable fusions with mouse myeloma lines. It would 10 be possible, however~ to use human, mouse, rabbit, sheep, goat or other cells instead.
Specialized myeloma cell lines have been developed from lymphocytic tumors for use in hyridoma-producing fusion - procedures [Kohler and Milstein, Ew. J. Immunol.6:511 (1976);
15 Shulman etal.,Nature276:269 (1978); Volk etal.,J. Virol.42:220 (1982)].~ These cell lines have been developed for at least three reasons. The first is to facilitate the selection of fused hybridomas from unfused and similarly indefinitely self-propagating myeloma cells. Usually, this is accomplished by 20 ~ using myelomas with enzyme deficiencies that render them - _ incapable of growing in certain selective media that support the growth of hybridomas. The second reason~arises~ from the inherent ability of lymphocytic tumor cells to produce their own antibodies. The purpose of using monoc!onal techniques is to -25 obtain fused hybrid cell lines wi~h unlimited iife-- spans that produce the desired single antibody under the genetic control of the somatic cell component of the hybridoma. To eliminate the production of tumor cell antibodies by the h~bridomas, myeloma cell lines incapable of producing endogenous light or heavy 30 immunoglobulin chains are used. A third re~son for selection of these cell lines is for their suitability and~ effieiency for fusion.

wo 93/22340 2 1 3 4 6 5 ~ Pcr/uss3/o3748 Many myeloma cell lines may be used for the produc~ion of fused cell hybrids, including, e.g., P3X63-Ag8, P3X63-AG8.653, P3/NS1-Ag4-1 (NS-l), Sp2/0-Agl4 and S194/5.XXO.Bu.1. The P3X63-Ag8 and NS-1 cell lines have been described by Kohler and Milstein [Eur. J. Imml~nol.6:511 (1976)].
Shulman et al. [Nature 276:269 (1978)] developed the Sp2/0-Agl4 myeloma line. The S194/5.XXO.Bu.1 line was reported by Trowbridge [J. Exp. Med. 148:313 (1979)].
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually involve mixing somatic cells with myeloma cells in a 10:1 proportion (although the proportion may vary from about 20:1 to about 1:1), respectively, in the presence of an agent or agents (chemical, viral or electrical) that promotes the fusion of cell membranes. Fusion methods have been described by Kohler and Milstein, supra, Gefter et al. [Somatic Cell Gene~. 3:231 (1977)], ¦;
and Volk e~ al. (J. Virol.-42:220 (1982)]. The fusion-promoting agents used by those investigators were Sendai virus and l `
polyethylene glycol (PEG). ~l Because fusion procedures produce viable hybrids at -very low frequency (e.g., when spleens are used as a source of somadc cells,~only one- hybrid is obtained for roughly every 1 x 105 spleen cells), it is essendal to have a means of selecting the `
fused cell hybnds- f~om the remaining unfused cells, particularly ~5 the unfused myeloma - cells. A means of detecting the desired antibody-producing- hybridomas among other resulting fused cell hybrids is also necessary. `
Generally, the sslection of fused cell hybrids is accomplished - by_ culturing the cells in media that support the -growth of hyb domas but prevent the growth of the unfused myeloma cells, which nonnally would go on dividing inde~mitely. .
The somatic cells used in the fusion do not maintain long-term viability in in vi~ro culture and hence do not pose a problem. For 21.~ 1fi'j~ .
WO 93/22340 PCI'/US93/03748 example, myeloma cells lacking hypoxanthine phosphoribosyl transferase (HPRT-negative) can be used. Selection against these cells is made in hypoxanthine/aminopterinlthymidine (HAT) medium, a medium in which the fused cell hybrids survive due to the HPRT-positive genotype of the spleen cells. The use of myeloma cells with different genetic ~eficiencies (drug sensitivities, etc.) that can be selected against in media supporting the growth of genotypically competent hybrids is also possible.
Several weeks are required to selectively culture the fused cell hybrids. Early in this time period, it is necessary to identify those hybrids which produce the desired antibody, so that they may subsequently be cloned and propagated.
Generally, around 10% of the hybrids obtaine~ produce the 15 desired antibody, although a range of from about 1 to about 30%
is not uncommon. The detection of antibody-producing hybrids can be achieved by any one of several standard assay methods, including en~yme-linked immunoassay and radioimmunoassay techniques which have been described in the literature [see, e.g., 20 Kennet et al. (editors), Monoclonal Antibodies and Hybridomas: A
New Dimension in Biological Analyses, pp. 376-384, Plenum Press, New York (1980)].
Once the desired fused cell hybrids have been selected and cloned into individual antibody-~roducing cell lines, 25 each cell line may be propagated in either~of-t~o standard ways.
A suspension of the hybridoma cells can be injected into a histocompatible animal. The injected animal will then develop tumors that secrete the specific monoc~on~l antibody produced by the fused cell hybrid. The body fluids of the animal, such as 30 serum or ascites fluid, can be tapped- to--~rovide monoclonal antibodies in high concentration. Alternatively, the individual cell lines may be propagated in vitro in laboratory culture vessels. The culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by wo 93/22340 ~ 13 ~ 6 5 ~ PCI'/US93/0374~

decantation, filtration or centrifugation, and subsequently purified .
Hybridomas producing monoclonal antibodies that can inhibit TSP-CD36 interactions are preferably identified by a two-stage screening process. First~ hybridoma clonss producing ~:~
- antibodies against TSP generally are iden~fied by, e.g., ELISA, radioimmunoassay or gel electrophoresis/Western blotting, using `-TSP as the antigen. Secondly, clones thus identi~led are further analyzed for increased binding affinity for TSP in the presence of polypeptide P139-155 or another ligand that binds to TSP at the same site. As explained above, prior binding of polypeptide P139-155 to TSP causes induction of the site to which the inhibitory a~ibody ligands of the invention bind and about a five-fold increase in binding of the antibodies. Such increased -~
binding of antibodies to TSP in the presence of the polypeptide can readily be detected and quanti~led, e.g., as described in the Example below.
Once a hybridoma producing the desired monoclonal antibody is obtained, techniques can be used to produce -interspecific monoclonal antibodies wherein the binding region of one species is combined with a non-binding region of the andbody o~ another species ~Liu et al., Proc. Natl. Acad. Sci. USA
84:3439 (1987)]. For example, the CDRs from a rodent monoclonal antibody can be grafted onto a human antibody, thereby "humar~izing" the rodent antibody [Riechmann et al., `~
Nature 332:323 (1988)~. More particularly, the CDRs can be grafted into a human antibody vanable region with or without human constant-regions. Such methodology has been used, e.g., to humanize a mouse monoclonal antibody against the pS~ (Tac) subunit of--the human interleukin-2 receptor [Queen et al., Proc.
Natl. Acad. Sci. USA 86:10029 (1989)3.

wo 93/22340 2 ~ :~ 4 fi ~ 5 Pcr/U~93/03748 lg Alternatively, human morioclonal antibodies can be prepared using the antigen disclosed herein and metbods described by Banchereau et al. [Science 251:70 (1991)].
The present invention also encompasses antibody binding fragments such as Fab, F(ab')2, Fv fragments, etc. The use and generation of fragments of antibodies is well known, e.g., Fab fragments ~Tijssen, Practice ~nd Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985~]~ Fv fragments [Hochman etal.,Biochemistryl2:1130 (1973); Sharon etal., 10 Biochemistry 15:1591 (1976); Ehrlich et al., U.S. Patent No.
4,355,023] and antibody half molecules (Auditore-Hargreaves, U.S. Patent No. 4,470,92~).
Human TSP for use in producing the hybridomas and mono~lonal antibodies of the invention can be prepared and 1~ purified by hlown methods, e.g., as described in the Example below.
Ligands other than polypeptides and antibodies or antibody fragments, such as cyclic peptides and non-peptide organic compounds, can be identified using the methods 20 disclosed herein. For example, other ligands that are functionally - equivalent to polypeptide P139-155 can be identified because they too, by binding to ~he same region on- TSP, will augment TSP-CD36 interactions and enhance the binding of polypeptide P93-110 to TSP. Similarly, other ligan~s that: are~functionally 25 equivalent to polypeptide P93-110 and the -antibodies produced by hybridoma clone 7AIe can be identified because tbeir binding to TSP will also be enhanced by prior binding of polypeptide P139-155, and they will inhibit TSP-CD36 interactions.
The ligands of the in~Jenti~_can be administered to a 30 human patient requiring modulation of-- th~ interaction of TSP
wi~h platelets or cells bearing TSP receptors either directly in a buffered physiological solution or in~the folln of a pharmaceutical composidon. Phatmaceutical compositions can wo93J~2340 ~,13465S Pcr/US93/03748 be prepared which contain effective amounts of one or more of -the ligands and a physiologically acceptable carrier. Such carriers are well known to ehose skilled in the art (see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: ;5 National Formulary, 1984, Mack Publishing Company, Easton, PA).
Administration may be by any suitable method, e.g., by parenteral administradon, including intravenous~
intraarterial, intraperitoneal, intramuscular and subcutaneous 10 administration. Administration may be by bolus injection, continuous infusion, sustained release from implanted delivery systems [Ur~uhart et al., Ann. Re,f. Pharmacol. Toxicol. 24:199 : `
(1984)] or by other known methods. -In general, the polypeptide and antibody ligands will 15 be administered at a dosage of from about 1 ~lg/kilogram body weightlday to about 50 mg/kilogram body weight/day1 with a preferred dosage of from about 1 mg/kilogram body weight/day I -to about 10 mg/~ilogram body weight/day; I ~
Determination of the proper dosage of a ligand for a ! `
20 particular situation is within the skill of the art. Generally, treatment is inithted with smaller dosages that are less than - ¦
opdmum. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For-conv-emence, the total daily dosage may be divided 25 and administered in portions during the day if desired.
The amount and frequency of administration of the ligands of- the~ invention and the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending_climcian, taking into account such factors as age, 30 conditio~ a-n~-size of the patient and severity of the condition(s) being treated.

WO93/22340 213~5 j j PCr/US93/03748 - EXAMPLI~
In the following non-limiting Example used for the purpose of illustration, percentages for solids in solid mixtures, liquids in liquids and solids in liquids are expressed on a wtlwt, vol/vol and wt/vol basis, respectively, unless otherwise indicated.
Polvpeptide SYnthesis The polypeptide ligands having amino acid sequences de~lned by SEQ ID NO: 1 and SEQ ID NO: 2 will for convenience be referred to below as polypeptides Pl39-lS5 and P93-l lO, respectively. These polypeptides, together with eight others having amino acid sequencei defimed by SEQ ID NOs: 3-lO, had sequences based upon subsequences of CD36.
All of the polypeptides were prepared by solid phase synthesis using an Applied Biosystems Model 430 peptide synthesizer and t-boc chemistry. Cleavage of the peptides from the resin was pe~formed with anhydrous HF at -5C in the presence of 10% anisole. The peptides were precipitated with ether, dissolved in 0.25 M acetic acid and lyophilized.
.
A cysteine and a tyrosine-residue~ were added to each peptide if the CD36 subse~uence upon which the peptide was based did not contain these residues, to allow the peptides to be - con3ugated to carner proteins or iodinated if- desired. The locations of these residues within the - peptides to which they were added were as follows:
i ..
-- -- - _ wo 93/22340 ~ 1 3 ~ ~ 5 5 22 PCr/US9310374X

~tide SEO ID NQ Re$idue (Numbe~
Cys (1) 2 ~ys (19) 3 Tyr (1); Cys (20) 4 Cys (l); Tyr (2) Cys (1) 6 ~ys(1) -8 Cys (l~ -9 Cys (l); ~yr (2~
Cys (l) ;
Collectively, the synthesized peptides comprised --about 40% of the exaacellular domain of CD36. ~
Protein Purification ;;
CD36 was pllrified from Triton X-l 14-solubilized human platelet membrane extracts by FPL(' anion exehange ' ~
chromatogIaphy essentially as deseribed by McGregor et al. :-~,J. Biol. Chem. 264:501 (1989)~ except that wheat germ agglul:inin affinity chromatography was performed instead of PPLC gel ~lltration as the fillal purification step, as descnbed ~y Tandon e~ al. [J. Biol. Chem~ 264:7570 (1989)~. TSP was purified from thrombil~-induced human platelet releasates, essentially as describe~ ~y Leun~ et al. ~.J. Clin. Invest. 70:~42 (l982?] and Clezardin et ~1. [Eur. J. Biockem. 154:9~ (1986)].
Monoclonal -Antiboidv Prc?~uçtiQn A three-month old female Lewis rat was immunized intraperitoneally (i.p.) with a 0.2~ ml solution of lO0 puri~led human TSP,- emulsified with an equal volume of complete Freund's adjuvant. Three weeks ater the initial immunization, the rat was-boosted i.p. with 50 ~Lg of TSP emulsi~led with 30 ` incompléte- Freund's adjuvant. This booster was repeated three ;
weeks later. Three weeks thereafter, after confirming serum reactivity by ELISA on whole TSP, the rat was boosted again.

wo 93/22340 ~ 1 ~3 4 fi ~ i PC~/US93/03748 Three days after the final booster injection the rat was sac~i~lced, blood was collected, and the spleen was removed for fuslon.
Spleen cells were fused with mouse myeloma cellst P3X63-Ag8.653 (ATCC CRL 158û), in a 1:1 ratio using 5 polyethylene glycol (PEG). The cell suspension (3.5 x 105 cells/ml in HAT medium) was distributed into 40 96-well plates.
Six days after plating the fusion products, the HAT
medium was replaced with HT medium. Hybridoma supernatants were tested in a primary screen for tbeir ability to 10 bind to-hwnan TSP two, three and four wèeks after fusion. Cells in positive wells were then expanded, and the medium was replaced with RPMI medium containing penicillin/strep~omycin (Pen-Strep) and 10% fetal calf serum (l:CS).
The prim~ry screen was a standard ELISA in which 1~ plates having U-shaped wells were coated overnight at 4C with 50 ~1 of a 10 ~g/ml solution of TSP in lX TBS (Tris-buffered saline; 20 mM Tns, 0.15 M NaCl, pH 7.4) with 2% CaCl. The plates were washed twice with TBS containing 2% CaCl and 0.05%
TWEEN 20~9 tpolyoxyethylenesorbitan monolaurate), after which 20 100 ~Ll of hybridoma supernatant were added to each well and the plates were incubated at room temperature for two hours.
The wells were washed twice as described above, and 50 ~1 of a 1:1,000 dilution of alkali~e phosphatase-conjugated goat anti-rat IgG in TBS/CaCl were added to~each well and 2~ incubated for one hour at room temperature. The wells were again washed twice, and 50 ~1 of BIORAD alkaline phosphatase substrate solution (a buffered solution of p-nitrophenyl phosphate) were added to each well. After a 15 minute incubation at room temperature, the- plates were read at 405 nm.
30 About 200 hybridoma cell lines testing-~positive in the primary screen were thus identified.

wo 93~22340 2 1 3 ~ 6 5 ~ 24 pcr/us93/o3748 The positive hybridomas were then evaluated in a secondary ELISA screen. In this screen U-shaped wells were -coated overnight at 4C as described by Leung e~ al. ~J. Clin. ~:.
Invest. 70:542 (1982)] with 200 ~1 of 5 ~Lg/ml human TSP in bicarbonate coating buffer (15 mM Na2C03, 34.8 mM NaHCO3, pH 9.6~. The plates were then blocked with 5% bovine serum 1;
albumin in TBS for 30 minutes at room temperature, and the 1 wells were washed twice with TBS containing 2 mM CaC12 and -0.05% rWEEN 20'!9.
One hundred microliter aliquots of the hybridoma supernatants were combined with 100 1ll of 2X Tris/0.05% 1~1 TWEEN 200, with and without polypeptide P139-155 to a final concentration of 10 ~g/ml. The resulting solutions were added to ;
the washed wells, and the plates were incubated for four hours 1~ at 37C. After washing the wetls twice as described above, 200 ~Ll of a 1:500 dilution of alkaline phosphatase-conjugated !:
goat anti-rat IgG were added to each well. The plates were incubated far one hour at 37C, developed and then read as described above.
Cells were selected from the secondary screen which secreted- andbodies that bound at least about 4 times more to the TSP iIr the presence of polypeptide P139-155 than in the absence of the polypepdde. Three hybridoma cell lines were thus idendfied, one of which was designated 7A~
~ The hybridoma cell lines testing positive in the secondary screen were then subcloned by limiting dilution. This was accomplished by seeding a solution containing 360 cellstwell in RPMI/10% FCS/Pen-Strep into the top row of a microtiter plate, and then carrying out serial 2-fold dilutions down the plate.--~he-bottom row would have been predicted to have 0.25 cell/well on average, as a result of this procedure.

wo 93/22340 ~ 1 3 4 ~ 5 ~ PCr/US93/03748 After two weeks, wells containing single colonies (as determined by visual inspec~on at 4X and 1 0X) were expanded.
As a result of such cloning of hybridoma cell line 7A, three clones were isolated, two of which were desig~ated 7AIe and 5 7AIh.
Protein and Polypeptide Iodination TSP, CD36 and the polypeptides having amino acid sequences defined by SEQ ID NOs: 1, 2 and 5 were radiolabeled with 125I using the IODOBEAD~ method (Pierce Chemical Co.)9 10 essentially as described by McGregor e~al.~J. Biol. Chem.264:~01 (1989)]. The labeled peptides were separated from free 125I by SEPHADEX G-25~9 gel filtration chromatography (1.3 x 25 cm) and eluted with phosphate buffered saline (PBS) at a flow rate of 0.5 ml/minute.
1~ Immunoadsor~tion of Labeled CD36 and Polv~eptide P139-1~5 by Antibody OKM5 12sI-CD36 or 125I-P139-155 (1 x 106 cpm/sample) was incubated with 3 ,ug/sample of antibody OKM5 (Ortho Pharmaceuticals) or a control monoclonal antibody against an 20 unrelated antigen (designated antibody lF~) for 2 hours at room temperature in the presence or absence~of- 10- ~g/sample of the various synthetic polypeptides. Protein A SEPHAROSE~ (15 ~l/sample) that had been pre-washed in PBS ~rit~ 0.5% bovine serum albumin (BSA) was added, and after ~ ho~r of incubation 25 at room temperature, the SEPHAROSE~9 was washed three times with Tris-Tween buffer [20 m~I Tris-HCl, 0.05% TWEEN 20~), pH 7.4] and the bound immune complexes~were eluted by boiling with sodium dodecylsulfate polyacrylamide- gel electrophoresis (SDS-PAGE) sample buffer [Laemmli, Natu~227:680 (1970)].
The eluted immune complexes were analyzed by SDS-PAGE (Laemmli, supra) and aut~radiography, with e~ual ~mounts of counts per sample loaded in the gels. 7.5% SDS-PAGE

wo93/2~340 2~ 13465~ 26 PCr/US93/03748 gels were used except for analysis of the labeled polypeptide, where 20% gels were used. The bands in the gels were located ~-by staining with Coomassie blue.
The results showed that by itself antibody OKM5 immunoadsorbed l25I-CD36 and that only polypeptide P139-155 consistently blocked such immunoadsorption. This inhibitory effect was speci~lc, because the polypeptide did not block immunoadsorption of 125I-albumin by a monoclonal antibody against albumin.
10. The specific inhibition of CD36 immunoadsorption by OKM5 by P139-155 suggested that the polypeptide might represent part of the CD36 epitope recognized by the antibody.
Alternatively, it was possible that the polypeptide bound to CD36, thereby causing a conforrnational change which indirectly affected the epitope against which OKM5 ~as directed.
To determine which was the case, the capacity of OKMS to directly immunoadsorb 125I-P139-155 was investigated. The result was that OKM5 did in fact directly immunoadsorb the labeled P139-155 polypeptide but not other polypeptides, thereby ruling out an indirect, conformational effect on CD36 Th-is-specific immunoadsorption was inhibited by excess unlabeled- P139-155.
Binding of Shr~mbos~ondin_to Polypeptide P139-155 Because antibody OKM5 has been shown to bloclc interaction between TSP and CD36 and polypeptide P139-155 represents part of-the- OKM5 epitope, it was possible that the polypeptide represented part of the binding site on CD36 for TSP. t To determine whet~er this was so, ELISA was carned out essentially as~described by Leung et al. [J. Clin. Invest. 70:542 (1982)] using surface-immobilized P139-155, TSP and a monaclonal-anti~ody-against TSP designated antibody P1035 (a~ailable from AMAC).

WO 93~22340 PCI/US93/03748 ~13465~

Briefly, polype.ptide P139-155-- was coated onto micro~iter wells at a concentration of 10 ~lg/ml in bicarbonate coating buffer ~15 mM Na2C03, 34.8 mM NaHC03, pH 9.5) by incubating overnight at 4C. The wells were washed with 5 Tris-Tween buffer, and nonspecific sites were blocked with Tris-Tween/0.5% BSA. TSP (1, 2, 4 and 8 llg/ml) was added in Tris-Tween buffer and incubated for 3 hours at 37C. Binding of TSP to polypeptide P139-155 was monitored by the sequential addition of anti-TSP antibody P1035 (10 ~lg/ml) followed by goat 10 anti-mouse IgG conjugated with alkaline phosphatase (1:7,500) and enzyme substrate. A~sorbance at 405 nm was measured in a TITERTEK~9 plate reader.
It was found that TSP bound to the immobilized polypeptide in a dose-dependent, saturable manner, but not to 1~ several of the other synthetic polypeptides. To further confirm i the specificity of the binding of TSP to irnmobilized polypeptide Pl39-15~, increasing amounts of soluble P139-1~5 were added during a binding assay canied out as described above. It was thereby found that the Wnding of TSP to immobilized 20 polypeptide P139-155 was inhibited 90% by the addition of 80 llg/ml soluble P139-155.
The results thus suggested that-polypeptide - ~ Pl39-I55 represents part of the CD36 molecule involved in T~P-CD36 binding.
25 Augmentation and Inhibition of the Bindin~ - ~~ ~
of CD36 to Thrombos~ondin The synthetic polypeptides described above were examined for possible effects on the binding of l25I-CD36 to surface-immobilized TSP. Briefly, TSP (5 ~Lg/m1)~ was 30 immobilized on microtiter wells and increasin~ amounts of soluble 125I-CD36 (specific radioactivity ~ 600,000 cpm/,ug ) were added in the presence or absence of 20 ~g/ml of each of the polypeptides in a binding assay carried out essentially as wo93/22340 ~,~346 jrj PCI/US~3/03748 ~ ~

described above. The only effects on the binding of the labeled CD36 to the immobilized TSP were observed in the presence of polypeptides P139-155 and P93-110. The results are shown in Fig. 2.
Because, as described above, polypeptide P139-155 appears to represent the part of the CD36 molecule involved in TSP-CD36 binding, it would be expected that by binding to TSP, the polypeptide would block the site at which TSP binds to CD36 and ~hereby inhibit the binding of the two proteins. Yet the data of Fig. 2 (upper curve) show just the opposite effect. Polypeptide P139-155 instead augmented or enhanced the binding of CD36 to TSP.
The binding of 125I-CD36 to immobilized TSP was augmented by 68.2 + 7.9% (mean ~ SEM, n=15) in the presence of soluble polypeptide P139-155 at a concentration of 10 llg/ml.
Seven other control polypeptides had no significant effect on -CD36 binding (n=12). This result was repeatedly observed, in five separate experiments.
In contrast, polypeptide P93-110 (Fig. 2, lower 20 curve), which a!so specifically binds to TSP but has no effect on the immunoprecipitation of CD-36 by antibody OKM5, did inhibit the binding of 125I-CD36 to immobilized TSP. The degree of ¦
inhibition was 58 + 2.5% (mean + SEM, n=9) in the presence of soluble polypepti~e~ Pg3-l:lO~at a concentration of S llg/ml and 25 69.7 + 5.7% (mean + SEM, n=18) at a concentration of 10 ~Lg/ml.
The results shown in Fig. 2 represent the average of triplicate determinafions obtained in one of two similar experiments.
Similar_inhibitory effects have been produced by the 30 monoclonal antibodies produced by hybridoma cell line 7A and subclones thereof such as 7AIe and. to a much lesser degree, J~13465S
wo 93~22340 PCr/USg3/0374X

7AIh. These two anti~odies, bound to different sites on TSP and were shown to vary substantially in inhibitory potency.
In binding studies carried out as described above, it was found that antibodies from subclone 7AIe in the presence of 5 polypeptide P139-155 significantly augmented the b;nding of 125I-CD36 to TSP. In the presence of the polypep~de~ 11.06 +
0.85 ng/well of the labeled CD36 bound; in the absence of the polypeptide, only 4.36 + 2.34 ng/well bound ~meaniSD, n=6).
In the absence of polypeptide P139-155, the 10 ~ndbodies from subclones 7AIe and 7AIh both partially inhibited the binding of 12~I-CD36 to TSP. When the polypep~de was present, however, the marked inhibition of binding seen with antibodies from subclone 7AIe was not produced by antibodies from subclone 7AIh.

These results indicate that after induction of the high affinity CD36 binding site on TSP by polypeptide P139-155, the enhanced binding and occupancy of the epitope by 7AIe andbodies blocked high affinity binding of TSP for CD36. This suggests that the 7AIe epitope is critically involved in the high 20 affinity binding site. In contrast, 7AIh antibodies evidently ~ bound to a different site on TSP which paltially interfered with baseline (without P139-1~5) binding, probably due to steric hindra~ce. This partial blockage, however, was completely eliminated in the presence of polypeptide P~g-~5~, with - 25 induction of the high affinity binding site. - ~
Effec~s On Platelet Aegre~ation To determine whether the contrasting effects of polypeptides P93-110 and P139-155 on the bind~ng of CD36 to TSP could be observed in a more physiologic~ system, the 30 polypeptides were examined for effects on human platelets.

wo 93/22340 Pcr/U~93/03748 ~1 3~ GS5 - Citrated platelet-rich human plasma was prepared and then incubated with polypeptides P139-155 and P93-l 10 at 37C and at 1,000 rpm in a Chrono-Log two-channel aggregometer as described by Leung [J. Clin. Invest. 74:1764 5 (1984)]. The platelets were then activated by the addition of ADP or collagen (Chrono-Log Corp., Havertown, PA), and aggregation was monitored as increased light transmission.
The results of a representative experiment are shown in Fig. 3, in which it can be seerl that polypeptide P139-155 at a 10 concentration of 30 ~lg/ml augmented platelet aggregation induced by 5 ~M ADP (Panel A) and 1.2 llgiml collagen (Panel B).
Polypeptide P93-110 at a concentration of 25 ~lg/ml partially inhibited platelet aggregation induced by 1.4 ~g/ml collagen ~`
(Panel C). Polypeptides designated P35-53, P208-224 and 1~ P313-329 having amino acid sequences def;ned by SEQ ID NOs: 3, 1-5 and 7, respectively, served as eontrols. The observed results were quantified by determining the areas under the aggregation tracings lby planimetry.
As shown in Panel A, polypeptide Pl39-1~5 did not 20 induce spontaneous platelet aggregation in the platelet-rich plasma but augmented aggregation induced by ADP by 44.5 +
10.7% (mean + Sl~M-, n=l~). This polypeptide also augmented collagen-induced aggegation by 101 + 17.3% (mean + SEM, n=5), ¦
with an increase in the size_of the platelet aggregates as -~
2~ evidenced by increasèd--amplitude in the aggregation tracing and by direct visual examination, compared to con~ol P3~-53 (Panel B). In contrast, as shown in Panel C, polypep~de P93-110 produced an inhibitor-y- effect on collagen-induced platelet aggregation, reducing - aggregation by 71.2 + 10.7%
30 (mean + SEM, n=7). _ Similar inhibitory effects have been produced by the monoclonal antibodies produced by hybridoma cell line 7A and subclone 7Ale. Data produced as described above using ~13~6~5 W O 93/22340 P ~ /~S93/03748 antibodies from subclone 7AIe instead of the polypeptides are shown in Fig. 4, in which the amount of antibodies used is shown to the right of each tracing.
As is evident from Fig. 4, increasing arnounts of 5 antibodies from subclone 7AIe produced concentration-dependent increasing inhibition of platelet aggregation. In contrast, antibodies from subclone 7AIh had little effect, even at a concen~ation at which 7AIe antibodies produced almost complete inhibition.
10 Induction of PolYpeptide P93-l lO Bindin~ to Thrombospondin To further investigate the effects of the P139-155 polypeptide on TSP, the binding of labeled polypeptide P93- 11 0 to immobilized TSP was measured, both in the presence and absence of polypeptide P139-155.
1~ This was accomplished by immobilizing TSP on microtiter wells as described above and then adding increasing concentrations of 125I-P93-110 (specific radioactivity 49,000 cpm/llg) in the presence or absence of a 4 ~g/ml roncentration of polypeptide P139-155 or of control polypeptide P208-224 or 20 P228-242 (having amino acid sequences defined ~y SEQ ID NOs:
S and 6, respectively). The plate was incubated fo-r 3 hours- at 37C. After washing three times with Tris-Tween buffer, the bound 125I-P93-110 was solubilized by SDS and measured-by gamma counting, with ~he results shown in Fig. 5. ~~
As shown in Fig. 5, 125I-P93-110 by itself did not bind appreciably to the immobilized TSP. In the presence of unlabeled polypeptide P139-155, however, there was a marked enhancement of 125I-P93-110 binding. This augmentation in binding by P139-155 was specific, because the- con~rol~
polypeplides at the same concentration had little or no effect.

WO 93/22340 ~ 1 ~ 4 6 ~i 5 PCr/US93/03748 - Furtber binding studies showed that 7AIe antibodies completely blocl~ed the binding of 1 25I-labeled polypeptide P93-110 to TSP in the presence of polypeptide P139-155. In contrast, 7AIh antib~dies showed no such blocking effece, again ;
5 suggesting that these antibodies did not bind directly to the critical high affinity binding site on TSP.
Taken together, the foregoing results suggest that the binding sites on TSP for 7AIe antibodies and polypeptide P~3-110 may be identical, or sterically related to each other. ~
~çntification of the Induced TSP Binding Site ~;
As noted above, the sequence Ser-Val-Thr-Cys-Gly (see also SEQ ID N0: 14) has been identified as a cell adhesive motif in TSP having homology to the maralia circumsporozoite protein. To determine whether this motif sequence was in~olved 1~ in the high af~mity CD36 binding site induced by the binding of polypeptide P139-155 to TSP, a pol~peptide having an amino acid sequence corresponding to that of residues 487 to 498 of the known sequence of TSP which contained the ~notif sequence `
was synthesized as described above, together with two 20 polypeptides containing the motif amino acid residues but in scrambled order. The amino acid soquences of these polypeptides are defined in the Sequence Listing by SEQ ID NOs:
11 (correct sequence) and 12 and 13 (scrambled sequences).
, _ ~Microtiter welrs--were coated with one of the 25 polypeptides as described- above, at a concentration of 10 llg/ml.
An unrelated control antibody or antibodies from subclone 7AIe or 7AIh were then added- at a concentration of 8 ~Lg/ml to the wells at 37C, and the plate was incubated for 3 hours. Following the incubadon, th=e ~hte was washed to remove unbound 30 andbodies, and the~bound antibodies were detected with goat anti-rat IgG conjugated with alkaline phosphatase. The results represented the~mean of two experiments, each done in triplicate.

WO 93/22340 2 1 3 4 6 ~ 5 PCI'~US93/03748 It was found that antibodies f~om subclone 7AIe bound significantly to the polypeptide containing the motif sequence, compared to the scrambled polypeptides (~A40s/min=12.64 x 10-3 vs 4.76 - 4.96 x 10-3), while antibodies 5 from subclone 7AIh and the unrelated control antibody showed little binding (~A40s/min=4.09 - 4.95 x 10-3).
Binding studies carried out as described above showed that 125I-labeled polypeptide P93-110 bound specifically to the immobilized motif-containing polypeptide 10 (9.89 ng/well) but not to the immobilized scrambled polypeptides ~2.41-2.54 ng/well). In contrast, 125I-labeled polypeptide P139-1~5 did not bind specifically to any of the immobilized polypeptides.
In view of the foregoing results, it is likely that the 16 previously-identified motif sequence is located in the high affinity CD36 binding site induced by the binding of polypeptide P139^155 to TSP.
HYbridoma De~osits Hybridoma clones 7AIe and 7AIh were 2Q deposited April 29, 1992 with the American Type Culture Collection (ATCC), Rockville, MD, under Accession Nos. HB 11û33 and HB 11034, respectively. These deposits were made under the conditions provided by the ATCC's agreement for-Culture Deposit for Patent Purposes, which assures that tfie~deposits will 25 be made available to the U.S. Commissioner of Patents and Trademarks ~pursuant to 35 U.S.C. 122 and 37 C.F.R. 1.14 and will be made available to the public upon issue of a U.S. patent, and which re~uires that the deposits be maintained. Availability of the deposited clones is not to be construe=d as=~a license to 30 practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

wo 93/22340 Pcr/US93/03748 3~
213~6~S ~
Many modifications and variations of this invention can be made without departing from its spirit and scope, as will become apparent to those skilled in the art. The speci~lc embodiments described herein are offered by way of example ,-only, and the invention is to be limited only by the terms of the appended claims.

~~

-- -= .. .

W093/2234û ~ ~ 1 65~ PCr/US93/03748 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Howard, Russell J.
Leun~, Lawrence L.K.

(ii) TITLE OF INVFNTION: Modulation of Thrombospondin-CD36 Interactions (iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE ADDRESS:
15: i i ~ - (A) AD:DRESSEE: Schering-plough Corporation !;

(B) STREET: One Giralda Farms 20 - (C) CITY: Madison ~ ~ , (D) STATE: New Jersey (E) COUNTRY: USA ~ : :

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppydisk .
(B) COMPUTER: Apple Macintos~

(C) OPERATING SYSTEM: Macintosh 6Ø5 :

w o 93/22340 2 1 3 ~ 6 5 5 PC-rIUS93/03748 - (D) SOFr-WARE: Microsoft Word 4.00B

(vi~ CURRENT APPLICATION DATA:

(A) AP PLIC ATIO N N U M BE R:

(B) FILIN G DATE: ~

(C) CLJ~S SIFIC ATIO N: -:
(vii) P RIO R A P PLIC ATIO N D ATA: I
!
(A) APPLICATION NUMBER: 08/009,260 1~ (B) FILING DATE: 25-JAN-1993 (A) APPLICATION NUMBER: 07/876,287 (B) FILING DATE; 30-APR-1992 (viii~ ATTORNEY/AGENT INFORMATION~

(A) NAME: Dulak-, Norrnan C.

(B) P~EGISTRATION NUMBER: 31,608 :~
.
(C) R EFF RE3~CE~C~C KE~r-N U M 8 ER: D X0270K

(ix~ TELECOMMUNICAT!ON INFORMATION:
(A) TE~LEPHONE: 201-822-7375 .
_.
(B) TELEFA3~: 201-822-7039 (C) TELEX: 219165 WO 93/22340 ~ 1 3 4 6 S S PCI/US93/0374X

(2) INFORMATION FOR SEQ ID NO: 1:

(i~ SEQUENCE CHARACTE~ISTICS:

(A) LENGTI J: 18 amino acids (B) TYPE: amino acid 1~
~D) TOPOLOGY: linear (ii) MOLECULE TYPE: pep~ide 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ~:
Cy Asn Leu Ala Val Ala Ala Ala Ser H1~ Ile Tyr Gln Asn Gln Phe Val Gln 20 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

.
~A) LENGTH: 19 amino acids (B) TYPE: amino acid (D~ TOPOL:)GY: linear 3~ (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Tyr Arg Val Arg Phe Leu Ala Lys Glu Asn Val Thr Gln Asp Ala Glu _ 15 35 A~p Asn Cys W O 93/22340 PCT/US93~03748 (2) !NFORMATION FOR SEQ ID NO: 3: ~

(i) SEQUENCECHARACTERISTICS: : -(A) LENGTH: 20 amino acids (B) T~PE: amino acid (D) TOPOLOGY: lin~ar (ii) MOLECULE lYPE: peptide 15 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Tyr Gln Lys Thr Ile Lys Ly~ Gln Val Val Leu (;lu Glu Gly Thr Ile Ala Phe Lys Cy5 ~ :~

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids ~B) TYPE: amino acid (D) TOPOLOGY: linear 3~
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRliTlON: SEQ ID NO: 4:
Cys Tyr Ile A3n Ly Ser Ly-~ Ser Ser Met Phe ~ln Val Arg Thr Leu ~:
1 s lo 15 213~5S5 Arg Glu Leu (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQIJENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids (B) TYPE: amino acid (D) TC)POLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: !
Cys Ala Asp Gly Val Tyr Ly~ Val Phe Asn Gly Lys Asp Asn Ile Ser 5 10 . 15 Lys Val (2) INFORMATION FOR SEQ ID NO: 6:

~i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids :
_ .

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide .
(xi) SEQUENCE DESCRIPTION: SFQ ID NO: 6:
Cy~ Asp ~hr Tyr Lys Gly Lys Arg Asn Leu Ser Tyr Trp &lu Ser His 4 ~ 5 40 ., I . .
(2) !NFORMATION FOR SEQ ID NO: 7: :

(i) SEQUENCE CHARACTERISTICS: ~
~ ::
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ~ -,~
(ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
15 Cy~ Thr Glu Ly~ Ile Ile Ser Ly~ Asn Cy~ Thr Ser Tyr Gly Val Leu 5 lO 15 Asp I :

(2~ INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:

:
(A) LENGTH: 18 amino acid~ -.. I
(B) TYPE: amino acid ¦
(D) TOPOLOGY: linear ~ ^~

(ii) MOLECULETYPE: peptide - : ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: .
Cy-~ Lg-~ Glu Gly Arg Pro Val Tyr Ile Sèr Leu Pro Hi-~ Phe Leu Tyr . .

Ala Ser -wo 93/22340 ?, ~ 3 ~ fi S ~ PCr/US93/0~74B

(2) INFORMATION FOR SEQ ID NO: 9: - ~

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (x~ SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Cy~ Tyr Val Ser Glu Pro Ile Asp Gly Leu A- ~n Pro Aan Glu Glu Glu 1 5 10 lS
His Arg Thr (2) INFORMATION FOR SEQ ID NO: 10:
. ;
(i) SEQUENCE CHARACTERISTICS: ~ ' (A) LENGTH: 18 amino acids (B) TYPE: amino acid 2~ -(D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: - `- -Cy-~ Val hy~ Pro Ser Glu Lys Ile Gln Val Leu Ly~ Asn Leu Lys Arg . .
A~n Tyr WO 93/22340 PCI`/US93/0374B
6S~ 47 (2) INFORMATtON FOR SEQ ID NO~

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acid~

(B) TYPE: amino acid (D) TOPOLOGY: linear :~
(ii) MOLECVLE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO~
Ser Val Thr Cys Gly Gly Gly Val Gln Lys Arg Ser ~; 1 5 10 :;
(2) INFORMATION FOR SEQ ID NO: 12: ~:

ti) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D3 TOPOLOGY: linear (ii) MOLECULElYPE: peptide - "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
30 Lys Ser Gly Thr Arg Gly Gln Ser Gly Val Cy~ Val (2) INFORMATION FOR SEQ ID NO: 13`

~13~6~5 wo 93~22340 PCI /US93/03748 .

(i) SEQUENCE CHARACTERISTICS: ~ ~

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~xi) SEQULNCE DESCRIPTION: SEQ ID NO: 13:
Val Ile Asp Gly Ser Ile Cy-~ Arg Gly Thr Thr Val . .10 1~ (2) INFORMATION FOR SEQ ID NO: 14:

(i)SEQUENCECHARACTERlSTlCS:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear 25 (ii) MOLECULETYPE: peptide - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: - -Ser Val Thr Cys Gly ~ -- ' .
-

Claims (15)

WHAT IS CLAIMED IS:
1. A ligand which selectively binds to a region of thrombospondin that specifically binds to a polypeptide having an amino acid sequence defined by SEQ ID NO: 1.
2. A ligand which selectively binds to a region of thrombospondin, the presence of which region is induced by the binding to thrombospondin of a polypeptide having an amino acid sequence defined by SEQ ID NO: 1.
3. A method for augmenting thrombospondin-mediated effects comprising contacting thrombospondin in the presence of platelets or cells bearing receptors for thrombospondin with an effective amount of a ligand which selectively binds to a region of thrombospondin that specifically binds to a polypeptide having an amino acid sequence defined by SEQ ID NO: 1.
4. The method of claim 3 in which the thrombospondin is contacted in the presence of platelets.
5. A method for inhibiting thrombospondin-mediated effects comprising contacting thrombospondin in the presence of platelets or cells bearing receptors for thrombospondin with an effective amount of a ligand which selectively binds to a region of thrombospondin, the presence of which region is induced by the binding to thrombospondin of a polypeptide having an amino acid sequence defined by SEQ ID NO: 1.
6. The method of claim 5 in which the thrombospondin is contacted in the presence of platelets.
7. The ligand or method of any one of claims 1, 3 or 4 in which the ligand is a polypeptide.
8. The ligand or method of claim 7 in which the ligand is a polypepide having an amino acid sequence defined by SEQ ID NO: 1.
9. The ligand or method of any one of claims 2, 5 or 6 in which the ligand is a monoclonal antibody.
10. The ligand or method of claim 9 in which the ligand is a monoclonal antibody produced by hybridoma clone 7AIe that binds to a region of thrombospondin containing a amino acid sequence defined by SEQ ID NO: 14.
11. The ligand or method of any one of claims 2, 5 or 6 in which the ligand is a polypeptide.
12. The ligand or method of claim 11 in which the ligand is a polypeptide having an amino acid sequence defined by SEQ ID NO: 2.
13. A pharmaceutical composition comprising a ligand of either claim 1 or claim 2 and a pharmaceutically acceptable carrier.
14. A method for making a pharmaceutical composition comprising admixing a ligand of either claim 1 or claim 2 with a pharmaceutically acceptable carrier.
15. Hybridoma clones 7AIe and 7AIh, deposited with the American Type Culture Collection under Accession Nos. HB 11033 and HB 11034, respectively.
CA002134655A 1992-04-30 1993-04-28 Modulation of thrombospondin-cd36 interactions Abandoned CA2134655A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US87628792A 1992-04-30 1992-04-30
US07/876,287 1992-04-30
US926093A 1993-01-25 1993-01-25
US08/009,260 1993-01-25

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US5367059A (en) * 1992-05-14 1994-11-22 W. R. Grace & Co.-Conn. Cys-Ser-Val-Thr-Cys-Gly specific tumor cell adhesion receptor
AU5161296A (en) * 1995-04-21 1996-11-07 Allelix Biopharmaceuticals Inc. Anti-haemorrhagic peptides
GB9509957D0 (en) * 1995-05-17 1995-07-12 Khalil Nasreen Post-translational activation of tgf-›1 involving the tsp-1 receptor cd36
US5611334A (en) 1995-07-07 1997-03-18 Muchin Jerome D Nose dilator device
US6098616A (en) 1998-03-13 2000-08-08 Acutek International Non-linear nasal dilator
CN106317191B (en) * 2015-06-23 2019-09-17 首都医科大学 Thr-Val-Gly-Cys-Ser, synthesis, activity and application
CN106279362B (en) * 2015-06-23 2019-07-12 首都医科大学 Arg-Leu-Val-Cys-Val, synthesis, pharmacological activity and application
ITUB20152122A1 (en) 2015-07-13 2017-01-13 Roberto Avataneo Nasal dilator device.

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US5200397A (en) * 1990-02-22 1993-04-06 W. R. Grace & Co.-Conn. Use of peptide analogs of thrombospondin for the inhibition of angiogenic activity
KR100206524B1 (en) * 1990-07-13 1999-07-01 어니스트 엠. 해데드 Cd53 cell surface antigen and recombinant dna encoding the same
EP1069137A1 (en) * 1990-09-24 2001-01-17 W.R. Grace & Co.-Conn. Peptides having thrombospondin-like activity and their therapeutic use

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