AU645077B2 - Cloning and production of human von willebrand factor GPIb binding domain polypeptides and methods of using same - Google Patents

Description

WO 91/13093 PCrUS91/01416 1 CLONING AND PRODUCTION OF HUMAN VON WILLEBRAND FACTOR GPIb EINDING DOMAIN POLYPEPTIDES AND METHODS OF USING SAMB Background of the Invention This application is a continuation-in-part of U.S. Serial No. 487,767, filed March 2, 1990, the contents of which are hereby incorporated by reference into the present disclosure.

Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.

This invention relates to the cloning and production of human von Willebrand Factor analogs and methods of using such analogs.

Structural Features of von Willebrand Factor Von Willebrand Factor (vWF) is a large plasaa protein which is synthesized in the endothelial cells which form the inner surface lining of the blood vessel wall, and by megakarocytes, the precursor of platelets. Large amounts of vWF are found in platelet a-granules, whose contents are released into the blood upon platelet activation. Newly synthesized vWF in endothelial cells may enter the blood via two alternative pathways. Part is secreted constitutively into the blood, mainly as disulfide-linked dimers or small multimers of a 250,000 dalton subunit. Alternatively, part enters secretory organelles called Weibel-Palade bodies.

The vWF stored in Weibel-Palade bodies is highly multimeric, ranging in size from that of a dimer to multimers of 50 or WO 91/13093 PC/US91/01416 -2more subunits, and can be released from the cells by treatment with secretatogues, such as thrombin. The highly multimeric vWF is the most effective in promoting platelet adhesion.

The gene encoding vWF has been isolated and shown to be greater than 150 kb in length. It is composed of over exons. The vWF mRNA is approximately 9000 bases in length and encodes a pre-pro-vWF of 2813 amino acids. Residues 1- 22 form a processed leader sequence which presumably is cleaved after entry of the protein into the rough endoplasmic reticulum. The N-terminal portion of the provWF (741 amino acids) is the pro-peptide which is not present in mature vWF. This peptide is present in the blood and has been shown to be identical to a blood protein previously known as von Willebrand Antigen II (vW AgII).

The pro-peptide is essential for the multimerization of vWF.

Cells into which a vWF cDNA containing only mature vWF sequences have been introduced produce only dimers. No function is known for the propeptide/vW AgII.

DNA sequence analysis has demonstrated that the pro-vWF precursor is composed of repeated domain subunits. Four different domains have been identified. Mature vWF consists of three A type, three B type, and two C type domains.

There are also two complete and one partial D type domain.

The pro-peptide consists of two D type domains, leading to the speculation that it may have associated functions.

Mature vWF is a multivalent molecule which has binding sites for several proteins. One of the binding sites recognizes the platelet glycoprotein Ib (GPIb). Using proteolytic digests this site has been localized to the region between amino acid residues 449 and 728 of mature vWF. In addition, vWF has at least two collagen binding sites, at least two WO 91/13093 PCC/US91/01416 -3heparin binding sites, a Factor VIII binding site, and a RGD site which binds to the platelet GP IIb/IIIa receptor.

Involvement Of vWF In Platelet Adhesion To Subendothelium Evidence that vWF, and specifically, the binding of vWF to the platelet GPIb receptor, is essential for normal platelet adhesion, is based on both clinical observations and in vitro studies. Patients with the bleeding disorder von Willebrand Disease (vWD) have reduced levels of vWF or are completely lacking in vWF. Alternatively, they may have defective vWF. Another disorder, Bernard-Soulier Syndrome (BSS), is characterized by platelets lacking GPIb receptors.

The in vitro system which most closely approximates the environment of a damaged blood vessel consists of a perfusion chamber in which an everted blood vessel segment (rabbit aorta, human post-mortem renal artery, or the human umbilical artery) is exposed to flowing blood. After stripping off the layer of endothelial cells from the vessel, blood is allowed to flow through the chamber. The extent of platelet adhesion is estimated directly by morphometry or indirectly using radiolabeled platelets.

Blood from patients with VWD or BSS does not support platelet adhesion in this system while normal blood does, indicating the need for vWF and platelet GPTb. Moreover, addition of monoclonal antibodies to GPIb prevents platelet adhesion as well. The vWF-dependence of platelet adhesion is more pronounced under conditions of high shear rates, such as that present in arterial flow. Under conditions of low shear rates, platelet adhesion may be facilitated by other adhesion proteins, such as fibronectin. Possibly, the adhesive forces provided by these other proteins are not adequate to support adhesion at high shear forces, and vWF dependence becomes apparent. Also, the multimeric nature of WO 91/13093 PCFrIUS91/01416S -4the vWF may provide for a stronger bond by binding more sites on the platelet.

About 20% of patients from whom clots have been removed by angioplasty or by administration of tissue plasminogen activator (tPA) suffer re-occlusion. This is presumably the result of damage to the endothelium during the treatment which results in the adhesion of platelets to the affected region on the inner surface of the vessel. This is followed by the aggregation of many layers of platelets and fibrin onto the previously adhered platelets, forming a thrombus.

To date none of the anti-platelet aggregation agents described in the literature prevent the initial platelet adhesion to the exposed sub-endothelium thereby preventing subsequent clot formation.

The subject invention provides non-glycosylated, biologically active polypeptides which comprise the vWF (von Willet. nd Factor) GPlb binding domain. These polypeptides may be used inter alia to inhibit platelet adhesion and aggregation in the treatment of subjects with conditions such as cerebrovascular disorders and cardiovascular disorders. This invention also provides expression plasmids encoding these polypeptides as well as methods of producing by transforming a bacterial cell and recovering such polypeptides. In addition, the subject invention provides methods of treating and preventing cerebrovascular, cardiovascular and other disorders using these polypeptides to inhibit platelet aggregation.

WO 91/1i093 PCT/US91/01416 ammary of the Invention This invention provides a non-glycosylated, biologically active polypeptide having the amino acid sequence: X-A-[Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Phe Val Val Trp Val Arg Ala Tyr lie Arg Arg lie Ala Ser Thr Phe Ser Lys Leu Leu Met Phe Val Arg Val lie Pro lie Arg Leu Val Leu Ser lie Val Ser Leu Ser Glu Ala Glu Phe Glu Val Leu Lys Asp Met Met Glu Arg Leu Arg Ile Ser Gin Val Ala Val Val Glu Tyr His Asp Gly Ser Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Ala Ser Gln Val Lys Tyr Ala Gly Ser Gin Ser Glu Val Leu Lys Tyr Thr Leu Phe Gin Ile Asp Arg Pro Glu Ala Ser Arg Ile Ala Ala Ser Gln Glu Pro Gin Arg Met Ser Arg Tyr Val Gin Gly Leu Lys Lys Lys Lys Val Val Gly lie Gly Pro His Ala Asn Leu Lys Ile Glu Lys Gin Ala Pro Glu Asn Lys Ala Ser Val Asp Glu Leu Glu Gln Gin Arg Asp Tyr Leu Cys]-B-COOH Ala Lys His Leu Val Ile Leu Asn Ile Gln Phe Glu wherein X is NH 2 -metiionine- or NH 2 A is a sequence of at least 1, but less than 35 amino acids, which sequence is present in naturally occurring vWF, the carboxy terminal amino acid of which is the tyrosine #508 shown in Figure 12; B is a sequence of at least 1, but less than 211 amino acids, which sequence is present in naturally occurring vWF, the amino terminal amino acid of which is the asparti3 acid #696 shown in Figure 12; and the two cysteines included within the bracketed sequence are joined by a disulfide bond.

WO 91/13093 PCr/IUS1/01416 -6- In addition, the subject invention provides a method of producing any of the above-described polypeptides which comprises transforming a bacterial cell with an expression plasmid encoding the polypeptide, culturing the resulting bacterial cell so that the cell produces the polypeptide encoded by the plasmid, and recovering the polypeptide so produced.

Furthermore, the subject invention provides a pharmaceutical composition comprising an amount of any of the abovedescribed polypeptides effective to inhibit platelet aggregation and a pharmaceutically acceptable carrier. The subject invention also provides a method of inhibiting platelet aggregation which comprises contacting platelets with an amount of any of the above-described polypeptides effective to inhibit platelet aggregation. In addition, the subject invention provides methods of treating, preventing or inhibiting disorders such as cerebrovascular or cardiovascular disorders or thrombosis, comprising administering to the'subject an amount of any of the abovedescribed polypeptides effective to treat or prevent such disorders.

The subject invention also provides a method for recovering a purified, biologically active above-described polypeptide which comprises: producing in a bacterial cell a first polypeptide having the amino acid sequence of the polypeptide but lacking the disulfide bond; disrupting the bacterial cell so as to produce a lysate containing the first polypeptide; WO 91/13093 PIP~/US91/01416 -7treating the lysate so as to obtain inclusion bodies containing the first polypeptide; contacting the inclusion bodies from step so as to obtain the first polypeptide in soluble form; treating the resulting first polypeptide so as to form the biologically active polypeptide; recovering the biologically active polypeptide so formed; and purifying the biologically active polypeptide so recovered.

WO 91/13093 PCrUS91/014166 -8- Brief Description of the Figures Figure 1: Construction of pvW1P This figure shows the construction of plasmid pvW1P. A series of vWF cDNA clones in A gt11 (isolated from a human endothelial cell cDNA library) were isolated. One cDNA clone covering the entire GPIb binding domain was subcloned into the EcoRI site of pUC19. The resulting plasmid, pvW1P, contains a 2.5 kb cDNA insert.

Picure 2: Construction of pvWF-VA1 This figure shows the construction of plasmid pvWF-VAl. A synthetic oligomer containing an ATG initiation codon located before the amino acid glu-437 the 437th amino acid in the vWF protein shown in Figure 12) was ligated to plasmid pvW1P digested with NdeI and Bsu36I. The resulting plasmid was designated pvWF-VA1, and has been deposited in coli strain S0930 under ATCC Accession No. 68530.

FiLSur 3: Construction of DvWF-VB This figure shows the construction of plasmid pvWF-VB1. A synthetic oligomer containing an ATG initiation codon located before the amino acid phe-443 (see Figure 12) was ligated to plasmid pvWlp digested with NdeI and Bsu36I. The resulting plasmid was designated pvWF-VB1.

Figure 4: Construction of pvWF-VA2 This figure shows the construction of plasmid pvWF-VA2. A synthetic oligomer containing a TAA termination codon located after the amino acid lys-728 (see Figure 12) was ligated to plasmid pvWF-VA1 digested with HindIII and Xmal.

WO 91/13093 PCr/US91/01416 -9- The resulting plasmid was designated pvWF-VA2.

Figure 5: Construction of pvWF-VB2 This figure shows the construction of plasmid pvWF-VB2. A synthetic oligomer containing a TAA termination codon was ligated to plasmid pvWF-VB1 digested with HindIII and XmaI.

The resulting plasmid was designated pvWF-VB2.

Figure 6: Construction of nvWF-VA3 This figure shows the construction of plasmid pvWF-VA3. An NdeI-EcoRV fragment was isolated from plasmid pvWF-VA2 and ligated to plasmid pMF-945 (constructed as described in Figure 11) digested with Ndel and PvuII. The plasmid obtained was designated pvWF-VA3. The plasmid expresses VA, a vWF GPIb binding domain polypeptide which includes aminc acids 437 to 728 (see Figure 12) under the control of the deo PIP 2 promoter.

gqure 7: Construction of DvWF-VB3 This figure shows the construction of nlasmid pvWF-VB3. An NdeI-EcoRV fragment was isolated from plasmid pvWF-VB2 and ligated to plasmid pMF-945 digested with Ndel and PvuII.

The plasmid obtained was designated pvWF-VB3. The plasmid expresses VB, a vWF GPIb binding domain polypeptide which includes amino acids 443 to 728 under the control of the deo

P

1

P

2 promoter.

Figure 8: Construction of pvWF-VC3 This figure shows the construction of plasmid pvWF-VC3. A synthetic linker was ligated to pvWF-VA3 digested with NdeI and TthlllI. The plasmid obtained was.designated pvWF-VC3, WO 91/13093 PCF/US91/01416 and has been deposited with the ATCC under ATCC Accession No. 68241. The plasmid expresses VC (also referred to as VCL or VC3), a vWF GPIb binding domain polypeptide which includes amino acids 504 to 728 (see Figure 12) under the control of the deo PIP 2 promoter.

gic&~_r Construction of pvWF-VD3 This figure shows the construction of plar 4 pvWF-VD3. A synthetic linker was ligated to pvWF-VA3 digested with Ndel and TthlllI. The plasmid obtained was designated pvWF-VD3.

The plasmid expresses VD, a vWF GPIb binding domain polypeptide which includes amino acids 513 to 728 (see Figure 12) under the control of the deo PiP 2 promoter.

icura 10: Relative Alianment of Plasmids Expressing vWF- GPIb Binding Domain Polypeptides This figure shows the relative alignment of the plasmids expressing the VWF-GPIb binding domain polypeptides. Also shown on the top two lines are representations of the vWF cDNA and the location of the GPIb binding domain coding region within the cDNA.

?i±ure 3 Construction of Plasmid DMF-945 This figure shows the construction of plasmid pMF-945.

Plasmid pEFF-920 (in Escherichia coli S0930, ATCC Accession No. 67706) was cleaved with BglII and NdeI, and the large fragment was isolated. This fragment was ligated to the vaall 540 bp fragment produced by cleaving plasmid pMF-5534 (ATCC Accession No. 67703) with BglII and 'deI. This produces plasmid pMF-945 which harbors the PAR sequence and in 5' and 3' order the deo P 1

P

2 promoter sequences, the modified deo ribosomal binding site with an enhancer WO~ 91/13093 PCr/US91101a16 -11sequence, a pGH analog coding sequence and the TjT 2 transcription termination sequences. Plasmid pMF-945 is a high level expressor of pGH analog protein.

Figre 12: Translated cDNA Seguence of Mature Human vWF This figure which consists of Figures 12A, 12B, 12C, 12D, 12E, 12F, 12G and 12H shows the translated cDNA sequence of mature human von Willebrand Factor.

This sequence was compiled using the data disclosed by Verweij, et al.? EMBO Journal 1: 1839-1847 (1986) and Sadler, et al., Proc. Natl. Acad. Sci. a: 6394-6398 (1985). This nucleotide sequence commences with nucleotide number 2519 (where nucleotide 1 relates to the start of the coding sequence for the signal peptide) and terminates with nucleotide 8668, a total of 6150 nucleotides encoding mature vWF consisting of 2050 amino acids. The translated amino acid sequence commences with amino acid number 1 and terminates with amino acid number 2050. The corresponding nucleotide and amino acid designations are used throughout this application.

PFliHr 13: Translated Seauence of VC. the Q I_ Binding Domain PolvDeDtide Expre jd b Plasmids pvWF-VC3 and pvWF-VCL This figure shows the translated sequence of the von Willebrand Factor GPIb binding domain polypeptide expressed by plasmids pvWF-VC3 (ATCC Accession No. 68241) and pvWF-VCL (ATCC Accession No. 68242).

The first codon ATG encoding the translation initiation codon methionine has been added to the nucleotide sequence corresponding to nucleotides 4028 to 4702 of the sequence of WO 91/13093 PCFUS91/01416 -12- Figure 12. This sequence encodes a rolypeptide containing 225 amino acids (plus the itiation methionine) corresponding to amino acid Leu 504 to amino acid Lys 728 of Figure 12, i.e. 226 amino acids in total.

Figure 14: Construction of DvWF-VCL This figure shows the construction of plasmid pvWF-VCL.

Plasmid pvWF-VC3 was digested with HindIII and Styl and the 860 base pair fragment isolated. This fragment was ligated with the large fragment isolated from .the HindIII-StyI digest of plasmid pMLK-100. The resulting plasmid was designated pvWF-VCL and deposited in E.coli 4300(F") with the ATCC under ATCC Accession No. 68242. This plasmid expresses VCL, the same vWF GPIb binding domain polypeptide as pvWF-VC3 (methionine plus amino acids 504-728), however under control of the APL promoter and the de ribosomal binding site.

Piquye 5: Construction of Plasmid pvWF-VE2 Plasmid pvWF-VA2 was digested with NdeI and PstI and the large fragment isolated. Synthetic oligomers No. 2 and No.

3 (shown in Figure 16) were treated with T4 polynucleotide kinase. The large pvWF-VA2 fragment was then ligated with synthetic oligomers No. 1 and No. 4, (shown in Figure 16) and with kinased oligomers No. 2 and No. 3. The resulting plasmid was designated pvWF-VE2.

Figure 16: Synthetic Oliaomers Used in Construction of pvWF-VE2.

This figure shows the four synthetic linkers (Nos. 1-4) used in construction of pvWF-VE2.

WO 91/13093 PC-r/US91/01416 -13rims 7 gonsruction of Elasmid ]2vwF-VE3 Plasmid pvWF-VE2 was digested with NdeI and HindIII and the small 770 bp fragment isolated and ligated with the large fragment isolated from the NdeI-HindIII digest of plasmid pMLK-7891. The resulting plasmid was designated pvWF-VE3.

Fiur I: Construction of amidl 1vWF-VEL Plasmid pvWF-VE3 was digested with XmnI, treated with bacterial alkaline phosphatase (BAP), and further digested with NdeI and HindIII. PlasmicpHLK-lO0 was digested with NdeI and HindIII and t::eated with BAP. The two digests were mixed and ligated, producing plasmid pvWF-VEL which expresses the DNA sequence corresponding to amino acids 469-728 of mature vWF under the control of the XP promoter and the cII ribosomal binding site.

Azu~re 19: The Effect of VCL on BTV-Induced acrcregation in Human Platelet Rich Plasma (PR)~ This f igure provides the results of a standardized von Willebrand Factor (vWF)-dependent aggregation assay using human PRP.

?i"p-2U: T2 Ef fect of VCL oQn B~JV-Indujced Aggregation This f igure provides the results of a standardized von Willebrand Factor (vWF)-dependent aggregation assay using rat PRP.

WO 91/13093 PCT/US91f01416 -14- Detailed DescriDtion of the Invention The plasmids pvWF-VC3, pvWF-VCL and pvWF-VA1 were deposited in Escherichia coli pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 68241, 68242 and 68530, respectively.

This invention provides a non-glycosylated, biologically active polypeptide having the amino acid sequence: X-A-[Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Phe Val Val Asp Met Met Trp Val Arg Val Ala Val Ala Tyr Ile Ily Leu Lys Arg Arg lie Ala Ser Gin Ala Ser Thr Ser Glu Val Phe Ser Lys Ile Asp Arg Leu Leu Met Ala Ser Gln Phe Val Arg Tyr Val Gin Val Ile Pro Val Gly Ile lie Arg Leu Ile Glu Lys Val Leu Ser Ser Val Asp Ala Glu Phe Glu Vl Glu Arg Leu Ar'g Ile Val Glu Tyr His Asp Asp Arg Lys Arg Pro Val Lys Tyr Ala Gly Leu Lys Tyr Thr Leu Pro Glu Ala Ser Arg Glu Pro Gin Arg Met Gly Leu Lys Lys Lys Gly Pro His Ala Asn Gin Ala Pro Glu Asn Glu Leu Glu Gin Gin Leu Lys Ala Ser Gin Lys Gly Ser His Ser Glu Leu Ser Gln Val Phe Gin Ile Ile Ala Leu Ser Arg Asn Lys Val Ile Leu Lys Gln Lys Ala Phe Arg Asp Glu Ile Val Ser Tyr Leu Cys]-B-COOH wherein X is NH 2 -methionine- or NH 2 A is a sequence of at least 1, but less than 35 amino acids, which sequence is present in naturally occurring human vWF, the carboxy terminal amino acid of which is the tyrosine #508 shown in Figure 12; WO 91/13093 PCr/US91/01416 B is a sequence of at least 1, but less than 211 amino acids, which sequence is present in naturally occurring human vWF, the amino terminal amino acid of which is the aspartic acid #696 shown in Figure 12; and the two cysteines included within the bracketed sequence are joined by a disulfide bond. The bracketed sequence comprises amino acids #509-1695 of Figure 12.

In one embodiment, this polypeptide has the amino acid sequence: X-[Leu His Asp Phe Tyr Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Glu Val Arg Ile His Asp Arg Pro Ala Gly Thr Leu Ser Arg Arg Met Lys Lys Ala Asn Glu Asn Gin Gin Pro Glu Val Thr Leu Asp Leu Lys Ser Gin Gly Ser ber Glu Ser Gin Phe Gin Ile Ala Ser Arg Lys Val Leu Lys Lys Ala Arg Asp Gly Ser Ser Ala Phe Val Lys Trp Val His Ala Tyr Le' Arg Arg Val Ala Ser lle Phe Ser Leu Leu Leu Asn Phe Val lie Val Ile Gin Ile Arg P',e Val Leu Glu Ile Val Arg Leu Ser Glu Ala Glu Phe Val Asp Met Met Glu Arg Leu Arg Val Ala Val Val Glu Tyr Ile Gly Leu Lys Asp Arg Lys Ile Ala Ser Gin Val Lys Tyr Thr Ser Glu Val Leu Lys Tyr Lys Ile Asp Arg Pro Glu Ala Met Ala Ser Gin Glu Pro Gin Arg Tyr Val Gln Gly Leu Lys Pro Val Gly Ile Gly Pro His Leu Ile Glu Lys Gin Ala Pro Ser Ser Val Asp Glu Leu Glu Ser Tyr Leu Cys Asp Leu Ala Leu Pro Pro Asp Met Ala Gin Leu Gly Val Ser Thr Leu Gly Ala Pro Pro Pro Thr Val Gly Pro Gly Leu Pro Lys]-COOH wherein X is NH 2 or NH 2 -methionine-, preferably

NH

2 -methionine-.

The bracketed sequence comprises amino acids #504-#728 of Figure 12.

WO 91/13093 PC/US91/01416 -16- One skilled in the art to which the subject invention pertains can readily make such polypeptides using recombinant or non-recombinant DNA techniques.

The polypeptides may be constructed using recombinant DNA technology. One means for obtaining the polypeptides is to express nucleic acid encoding the polypeptides in a suitable host, such as a bacterial, yeast, or mammalian cell, using methods well known in the art, and recovering the polypeptide after it has been expressed in such a host.

Examples of vectors that may be used to express the nucleic acid encoding the polypeptides are viruses such as bacteriophages (such as phage lambda), cosmids, plasmids, and other recombination vectors. Nucleic acid molecules are inserted into vector genomes by methods well known in the art. For example, using conventional restriction enzyme sites, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules which base pair with each other and are then ligated together with a ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA, which is then digested with the restriction enzyme which cuts at that site. Other means are also available.

Vectors comprising nucleic acid encoding the polypeptides may be adapted for expression in a bacterial cell, a yeast cell, or a mammalian cell which additionally comprise the regulatory elements necessary for axpresslon of the nucleic acid in the bacterial, yeast, or mammalian cells so located relative to the nucleic acid encoding the polypeptide as to permit expression thereof. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.

WOJ( 91/13093 PC/US91/01416 -17- For example, a bacterial expression vector may include a promoter such as the A PL or Lde promoters and for transcription initiation the CeI or ribosomal binding sites. Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art, for example the methods described above for constructing vectors in general.

In addition, non-recombinant techniques such as chemical synthesis, synthetic DNA or cDNA may be used to obtain the above-described polypeptides. One means of isolating the polypeptide is to probe a human genomic library with a natural or artificially designed DNA probe, using methods well known in the art. DNA and cDNA molecules which encode the polypeptides may be used to obtain complementary genomic DNA, cDNA or RNA from human, mammalian or other animal sources, or to isolate related cDNA or genomic clones by the screening of cDNA or genomic libraries.

The subject invention further provides a pharmaceutical composition comprising an amount of any of the abovedescribed polypeptides effective to inhibit platelet aggregation and a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers. Such carriers are well known in the art and may include, but are in no way and are not intended to be limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, and various types of wetting agents. Other carriers may also include sterile solutions, tablets, coated tablets, and capsules.

Typically such carriers contain excipients such as starch, WO 91/ 113093 PC/US91/01a16 -18milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods.

The composition has an amount sufficient to result in a blood concentration of 0.06 to 58 1M, preferably between about 0.06 and 29 pM, for example 0.23 to 23 MM. Expressed in different terms, the amount should be 0.1 to 100 mg/Kg body weight, preferably 0.1 to 50 mg/Kg body weight, for example 0.4 to 40 mg/kG body weight.

The administration of the composition may be effected by any of the well known methods, including but not limited to intravenous, intramuscular, subcutaneous and oral administration.

This invention also provides a method of inhibiting platelet aggregation which comprises contacting platelets with an amount of any of the above-described polypeptides effective to inhibit platelet aggregation so as to inhibit platelet aggregation.

This invention also provides expression plasmids encoding the above-described polypeptides. In one embodiment, the expression plasmid encoding the polypeptide with the bracketed sequence, i.e. amino acids #504-#728 of Figure 12, is designated pvWF-VC3 and is deposited under ATCC Accession No. 68241. In another embodiment, the expression plasmid encoding a polypeptide with the bracketed sequence, i.e.

amino acids #504-1728 of Figure 12, is designated pvWF-VCL and is deposited under ATCC Accession No. 68242.

WO 91/13093 PC;/US91/01416 -19- The expression plasmids of this invention further comprise suitable regulatory elements positioned within the plasmid relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell, such as promoter and operators, e.g. deo P 1

P

2 and A PLOL, ribosomal binding sites, e.g. !U and CII, and repressors.

Other suitable regulatory elements include, for example, the lac, trp, tac, and Ipp promoters (European Patent Application Publication No. 0303972, published February 22, 1989).

The suitable regulatory elements are positioned within the plasmid relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell. In preferred embodiments of the invention, the regulatory elements are positioned close to and upstream of the DNA encoding the polypeptide.

The expression plasmids of this invention may be introduced into suitable host cells, preferably bacterial host cells.

Preferred bacterial host cells are Escherichia coli cells.

Examples of suitable Escherichia coli cells are strains S0930 or 4300, but other Escherichia coli strains and other bacteria can also be used as host cells for the plasmids.

Such bacteria include Pseudomonas Deruginosa and Bacillus subtilis.

The bacteria used as hosts may be any strain including auxotrophic (such as A1645), prototrophic (such as A4255), and lytic strains; F and F- strains; strains harboring the c1 857 repressor sequence of the A prophage (such as A1645 and A4255); and strains deleted for the deo repressors and the deo gene (see European Patent Application Publication No. 0303972, published February 22, 1989). Escherichia coli strain A4255 (F has been deposited under ATCC Accession WO 91/13093 PC/US91/01416 No. 53468, and Escheriia coli strain A1645 has been deposited under ATCC Accession No. 67829.

The invention provides a bacterial cell which comprises these expression plasmids. In one embodiment, the bacterial cell is an Escherichia coli cell. In preferred embodiments, the invention provides an Escherichia coli cell containing the plasmid designated pvWF-VAl, deposited in E.u__li strain S0930 with the ATCC under ATCC Accession No. 68530; pvWF- VA3; pvWF-VB3; pvWF-VC3, deposited in EZcoli strain S0930 with the ATCC under ATCC Accession No. 68241; pvWF-VD3; or pvWF-VCL, deposited in E. colj strain 4300(F") with the ATCC under ATCC Accession No. 68242.

All the E. coli host strains described above can be "cured" of the plasmids they harbor by methods well-known in the art, e.g. the ethidium bromide method described by R.P.

Novick in Bacteriol. Review 33, 210 (1969).

In addition, the subject invention provides a method of producing any of the above-described polypeptides which comprises transforming a bacterial cell with an expression plasmid encoding the polypeptide, culturing the resulting bacterial cell so that the cell produces the polypeptide encoded by the plasmid, and recovering the polypeptide so produced.

Furthermore, the invention provides a method of treating a subject with a cerebrovascular disorder which comprises administering to the subject an amount of any of the polypeptides of the invention effective to inhibit platelet aggregation.

Also provided is a method of treating a subject with a cardiovascular disorder which comprises administering to the WO 91/113093 PC/US91/01416 -21subject an amount of a polypeptide effective to inhibit platelet aggregation. Examples of cardiovascular disorders susceptible to treatment include acute myocardial infarction or angina.

Further, the subject invention provides method of inhibiting platelet aggregation in a subject prior to, during, or after the subject has undergone angioplasty, thrombolytic treatment, or coronary bypass surgery which comprises administering to the subject an amount of a polypeptide of the invention effective to inhibit platelet aggregation.

The invention also provides a method of maintaining blood vessel patency in a subject prior to, during, or after the subject has undergone coronary bypass surgery, which comprises administering to the subject an amount of a polypeptide of the invention effective to inhibit platelet aggregation.

The invention also provides a method of treating a subject having cancer which comprises administering to the subject an amount of a polypeptide of the invention effective to retard tumor metastasis.

The invention also provides a method of inhibiting thrombosis in a subject which comprises administering to the subject an amount of a polypeptide of the invention effective to inhibit the thrombosis. The thrombosis may be associated with an inflammatory response.

In addition, the subject invention provides a polypeptide of the invention bound to a solid matrix.

The invention also provides a method of treating a subject suffering from platelet adhesion to damaged vascular W~rO 91/13093 PClT/US91/01416 surfaces which comprises administering to the subject an amount of the polypeptide of the invention effective to inhibit platelet adhesion to damaged vascular surfaces.

The invention also provides a method of preventing platelet adhesion to a prosthetic material or device in a subject which comprises administering to the subject an amount of the polypeptide of the invention effective to prevent platelet adhesion to the material or device.

The invention also provides a method of inhibiting reocclusion in a subject following angioplasty or thrombolysis which comprises administering to the subject an amount of the polypeptide of the invention effective to inhibit reocclusion.

The invention also provides a method of preventing vasoocclusive crises in a subject suffering from sickle cell anemia which comprises administering to the subject an amount of the polypeptide of the invention effective to prevent vaso-occlusive crises.

The invention also provides a method of preventing arteriosclerosis in a subject which comprises administering to the subject an amount of the polypeptide of the invention effective to prevent arteriosclerosis.

The invention also provides a method of thrombolytic treatment of thrombi-containing, platelet-rich aggregates in a subject which comprises administering to the subject an amount of the polypeptide of the invention effective to treat thrombi-containing, platelet-rich aggregates.

The invention also provides a method of preventing platelet activation and thrombus formation due to high shear forces WO 91/13093 PCT/US91/01416 in a subject suffering from stenosed or partially obstructed arteries which comprises administering to the subject an amount of the polypeptide of the invention effective to prevent platelet activation and thrombus formation.

The invention also provides a method of preventing thrombininduced platelet activation in a subject which comprises administering to the subject an amount of the polypeptide of the invention effective to prevent thrombin-induced platelet activation.

The invention also provides a method of preventing stenosis as a result of smooth muscle proliferation following vascular injury in a subject which comprises administering to the subject an amount of the polypeptide of the invention effective to prevent stenosis.

The invention also provides a method for recovering a purified, biologically active polypeptide of the invention which comprises: producing in a bacterial cell a first polypeptide having the amino acid sequence of the polypeptide but lacking the disulfide bond; disrupting the bacterial cell so as to produce a lysate containing the first polypeptide; treating the lysate so as to obtain inclusion bodies containing the first polypeptide; contacting the inclusion bodies from step WO 91/13093 PCT/US9'.Y1416 -24so as to obtain the first polypeptide in soluble form; treating the resulting first polypeptide so as to form the biologically active polypeptide; recovering the biologically active polypeptide so formed; and purifying the biologically active polypeptide so recovered.

Step may comprise contacting the polypeptide with d thiol-containing compound and disulfide so as to refold and reoxidize the polypeptide. Preferably, the thiol-containing compound is glutathione, thioredoxin, B-mercaptoethanol or cysteine.

The contacting of step may be effected in the presence of a denaturant such as guanidine hydrochloride or urea.

The recovery of the polypeptide in step may comprise removing the denaturant by dialysis.

In step the biologically active polyr -tide may be purified by cation exchange chromatography.

The first polypoptide may also be purified by cation exchange chromatography after step WO 91/13093 PCrUS9101416 The examples which follow are set forth to aid in understanding the invention but are not intended to, and should not be so construed as to, limit its scope in any way. The examples do not include detailed descriptions for conventional methods employed in the construction of vectors, the insertion of genes encoding polypeptides of interest into such vectors or the introduction of the resulting plasmids into bacterial hosts. Such methods are well-k .wn to those skilled in the art and are described in numerous publications including Sambrook, Fritsch and Maniatis, Molecular CloninG: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, USA, (1989).

All the references to map positions correspond to the identically numbered positions along the translated nucleotide sequence of mature human von Willebrand Factor shown in Figure 12.

Cxample 1: Cloning and Exression of vWF GPIb Binding Domain Polvypetides cDNA Cloning of Human vWF GPlb Binding Domain A human endothelial cDNA library (obtained from CLONTECH Laboratories, Inc.) in X gtll was screened for human vWF positive sequences using two synthetic DWA probes. The probes were synthesized according to the published DNA sequence (Sadler et al., Proc. Nat. Acad. Sci. Uj: 6394-8 (1985) and Verweij et al., EMBO J. 3: 1829-47 (1986)) of human vWF (flanking 5' end and 3' end of the vWF domain known to bind the GPIb receptor) (see Figure 12).

The synthetic probes have the following sequences: WO) 91/13093 PPr/JS91/01416 -26- Seouence Mcletidesj AAATCTGGCAGTGCTCAGGGTCACTGGGATTCAAGGTGAC 3863-3902 CCAGGACGAACGCCACATCCAGAACCATGGAGTTCCTCTT 4700-4739 A series of vWF cDNA clones covering the entire GPIb binding domain were identified and isolated. The cDNA fragments were subclned into EcoRI site of pUC-19 (New England Biolabs, Inc.). One of the subclones, designated pvWIP (Figure contains a 2.5 Kb insert. This 2.5 Kb insert covers the entire GPIb binding domain extending from 550 bp upstream of the GPIb binding site to 1100 bp downstream of the GPIb binding site. (The subclone pvW1P has also been designated pvWF-1P).

Manipulation of DNA Codina for the vWF GPIb Binding Domain In order to obtain expression of the GPIb binding domain in Escherichia coli under the regulation of the geo P 1

P

2 promoter, the cDNA fragment of vWF, derived from plasmid pvW1P was used for further manipulations as described below.

As indicated previously, the vWF tryptic digest fragment that binds the GPIb receptor is from amino acid Val 449 to amino acid Ly_ 728.

A. Subcloning of the 5' end of vWF GPIb binding domain and addition of a translation initiation codon ATG.

Plasmid pvW1P has two convenient restriction sites at the end. Bsu36I which cuts at the DNA sequence corresponding to amino acid Ser (445), and TthlllI which cuts at amino acid AsP (514). Synthetic fragments of various size were designed that insert an ATG translation initiation codon at the 5' end as well as additional amino acids. This was done first, in order to maximize the chances of obtaining high levels of expression. Second, they are a first step towards WO 91/13093 PCr/US91/01416 -27reducing the size of the vWF GPIb binding domain peptide down to the minimal size needed, possibly eliminating collagen and heparin binding sites which may ultimately interfere with the function of the product.

Al. Amino acid Qju 137 at 5' e2nd Synthetic oligomers with the sequences: T&TGAGGTGGCTGGCCGGCGTTTTGCC 3' 3' ACTCCACCGACCGGCCGCAAACa~gT 51 NdeI Bsu36I were ligated to plasmid pvWF-lP digested with NdeI and Bsu36I (see Figure The plasmid obtained was designated pvWF-VAl. Plasmid pvWF-VA1 has been maintained in strain S0930 and was deposited under ATCC Accession No. 68530.

A2. Amino acid Phe 443 at .1,P~ Synthetic oligomers with the sequences: 51 TAIMTTTGCC 31 31 ACAAACGGAGT were ligated to plasmid pvWlP digested with NdeI and Bsu36I (see Figure The plasmid obtained was designated pvWF-VB1.

WO 91/13093 PCF/US91/01416 -28- B. Subcloning of the 3' end o'f vWF GPIb binding domain, introduction of translation stop codon.

Bl. Introduction of stop codon in 2iasmid D2vWF-VAJ A synthetic oligomer with the sequence: *-CCGGGGCTCTTGGGGGTTTCGACCCTGGGGCCCAAGMGATATCA- 3' 3' -CCGAGAACCCCCAAAGCTGGGACCCCGGGTTCATTCTATAGTTCGA-51 was ligated to an XmaI and HindIII digested. plasmid pvWF-VAI (see Figure The plasmid obtained was designated pvWF-VA2. This newly constructed plasmid cont~ins a translation termination codon TAA adjacent to amino acid 728 (Lys) and EcoRV site.

B2. introduction of translation stop qZ in pDIsmid

YW-B

A synthetic oligomer with the sequence: 5'1- CCGGGGCTCTTGGGGTTTCGACCCTGGGGCCCAAGMGATATCA 31 3' CCGAGAACCCCAAAGCTGGGACCCCGGGTTCATTCTATAGTTCGA was ligated to plasmid pvWF-VBl digested with XmaI and HindIII. The plasmid obtained was designated pvWF-VB2 (see Figure Exi~ession of the vWF GP~b binding domain in Escherichi1A co 1 In order to obtain expression of the vWF GPIb binding domain various expression plasmids were constructed based on a dec WO 91/13093 PCF/US91/01416 -29- PlP2 constitutive promoter system.

1. Eairessign of a vWE GPIb bidngdmanDove~tide including amino acid Qlu 437 toQ amino0 r.i _s72-8 (baged on 1ASMid 1DVWF-VAZ) An NdeI-EcoRV fragment was isolated from plasmid pVWF-VA2 and ligated into plasmid pMF-945 (see Figure 11) digested with NdeI and PvuII (see Figure The plasmid obtained was designated as pvWF-VA3 and was maintained in Escherichia coli strain S0930.

2. Exlression of a vWF GPIb binding !domain3 ~leTtide jing udinr amino acid h 4 oaioacid- Lys72- (b-e on Plasmid DvWF-VB2) An NdeI-EcoRV fragment was isolated from plasmid pvWF-VB2 and ligated into plasmid pMF-945 digested with NdeI and PvuII (see Figure The plasmid obtained was designated as pvWF-VB3 and was maintained in eiiai go strain S0930.

3. ExtMressign of a MWF GPIb2 binding domain 2olv~ei2tide including amino acid Leu 504 to amino acid Lys 728 (based on eX~ression plasmid DvWF-VA3).

A synthetic oligomer with the sequence: TAIG-.GCACGATTTCTACTGCAGCAGGCTACTGGACC 3' 31 =ACAACGTGCTAAAGATGACGTCGTCCGATGACggA,,- 51 NdeI Tth1lII WO 91/13093 PCT/US91/01416 was ligated to plasmid pvWF-VA3 digested with WdeI and Tthlli. The plasmid obtained was designated as pvWF-VC3 (see Figure 8) Plasmid pvWF--VC3 is maintained in ZzQharigia goli. strain S0930 ipnd has been deposited with the ATCC under Accession No. 68241 (also see Figure 13).

4. Xxp~rgssion of a vWF GPIb2 binding domain DolvyDe2&tide including amnoaid Leu 513 to amino- acid Lys 728 (base4 on--expression plasmidDvvWF-YA3) A synthetic oligomer with the sequence: 51 TAMCTGGACC 3' ACGACCTl NdeI TthlllI was ligated to plasmid pvWF-VA3 digested with NdeI and TthlllI. The plasmid obtain~d was designated pvWF-VD3 (see Figure 9) -Plasmid pvWF-VD3 is maintained in Escherichia old strain S0930.

Expression of vWF-GPjb binding domain Rolv2entides The relative alignment of the expression plasmids is shown in Figure 10. Plasmids pvWF-VA3, pvWF-VB3, pvWF-VC3 and pvWF-VD3 in Eschgrichia coli strain S0930 were used in order to analyze the levels of expression of the, various vWF-GPIb binding domain peptides. The clones obtained were grown in LB medium containing Amp (100 gg/ml) at 37 0 C for 48 hours.

After 48 hours growth bacterial cells were harvested and centrifuged for 2 minutes at 10,000 RPM. Pellets were dissolved in 1/10 volume of 50 mM Tris-HC1 pH=8.0. Sample WO 91/13093 PC~3/US91/01416 -31buffer (containing SDS and B-mercaptoethanol) was added.

Samples were boiled for 10 minutes and loaded on a 10% SDS polyacrylamide gel. The expression of the vWF GPIb binding domain polypeptides in clones pvWF-VA3, pvWF-VB3 and pvWF- VD3 was low relative to the bacterial total proteins. The vWF polypeptides from these clones were detectable by Western blot analysis using commercially available polyclonal vWF antibody (Dekopatts a/s, Glostrup, Denmark).

However, clones originated from Escherichia coli strain S0930 transformed with plasmid pvWF-VC3 expressed the vWF GPIb binding domain polypeptide (amino acid Leu 504 to amino acid Lys 728 plus methionine) at high levels (as a major band) detectable upon Coumassie staining.

Escherichia coli strain S0930 harboring plasmid pvWF-VC3 was deposited with the ATCC under Accession No. 68241.

Subsequently, an inducible plasmid was constructed which contains the same vWF coding region as pvWF-VC3, expressed under the control of the A PL promoter and the deo ribosomal binding site (see Figure 14). This new plasmid, designated pvWF-VCL, proved to be a high expressor of VCL, the vWF GPIb binding domain polypeptide (methionine plus amino acid Leu 504 to amino acid Lys 728). This plasmid was deposited in Escherichia coli strain 4300 with the ATCC under Accession No. 68242. Escherichia coli strain 4300, constructed from Escherichia coli strain ATCC Accession No. 12435, is a wildtype, biotin dependent strain, harboring the A ci857 temperature-sensitive repressor. (A third plasmid construct harboring the same vWF coding region under the control of the promoter and the cll ribosomal binding site did not express any vWF peptide detectable by Coomassie staining.) The NdeI-HindIII insert of pvWF-VCL can be conveniently subcloned into other expression vectors such as commercially WO 91/13093 PCT/US91/01416 -32available pUC19 for production of a series of polypeptides which include the same amino acid sequence from amino acid 509 (cys) to amino acid 695 (cys) and have the same biological activity.

WO 9113093 PCFI/US91/01416 -33- Xampe Fermentation of Bacteria Ex-ressina vWF GPIb Binding Domain Polvpentides During scale-up fermentations of clone pvWF-VC3 it was found that the host tends to lose the plasmid due to instability.

The loss of plasmids caused a reduction in vWF GPIb binding domain polypeptide expression. It was found necessary to maintain continuous selective pressure continuous addition of Ampicillin) in order to maintain plasmid copy number and to maintain the expression levels. Large scale fermentation was carried out for 12 hours.

Fermentation was carried out in the following growth medium: N-Z amino AS 20 gr Yeast extract 10 gr NaCl 5 gr

K

2

HPO

4 2.5 gr MgSO 4 7H20 1.0 gr Anti foam 0.4 ml Fructose was added to the growth medium at final concentration of 150 ml/liter and Ampicillin (100 mg/ml) was pumped continuously into the fermentor (total of 8 ml/liter). Fermentation was carried out for 12 hours at 370C.

Purification of polvpeptides Cells were harvested after 12 hours fermentation and centrifuged. The bacterial pellet obtained was resuspended in buffer [50 mM Tris pH=8.0, 50 mM NaC1, 1 mM EDTA, 1mM DTT (dithiothreitol), 1 mM PMSF (Phenylmethylsulfonyl fluoride) and 10% Glycerol). After additional centrifugation and sonication the vWF GPIb binding domain polypeptide was found WOY 91/13093 IPCr/US91/01416 -34in the pellet.

The vWF GPIb binding domain polypeptide was further purified by solubilization of the pellet in 8M Urea containing 10 mM DTT, 25 mM Tris pH=8 and 1 mM EDTA at room temperature. The solubilized pellet was fractionated on a DEAE cellulose ion exchange column chromatography. (Elution buffer as above except 0.5 mM DTT).

The vWF GPIb binding domain polypeptide was eluted at 150 mM NaC1. After dilution to 50 mM NaC1 (in the above buffer) the partially purified peptide was loaded on a Q-Sepharose column. Elution from the Q-Sepharose column was carried out at various NaC1 concentrations (step elution). The vWF GPIb binding domain peptide was pooled in four peaks which aluted at 100 mM, 200 mM, 250 mM and 500 mM NaC1. All four peaks were dialyzed against 150 mM NaCI and 50 mM Tris pH=8 for 36 hours. During the dialysis the Urea concentration of the dialysis solution was reduced in a linear gradient from 6M Urea to no Urea.

WO 91/13093 PCFUS91/01416 11jjMR1 j: Biological Activitr of vWF GPIb Binding Domain PolyDeptides Platelet Aagregation Assays vWF prearation: Human plasma-derived vWF was purified from human outdated blood bank plasma according to J. Loscalzo and R.I. Handin, Biochemistry 2a: 3880-3886 (1984). The purified plasmaderived vWF was concentrated by Amicon 100,000 cut-off filter membrane, to a final concentration of 0.25 mg/ml.

Asialo-vWF preparation: The purified plasma-derived human vWF was desialyated according to L. DeMarco and S. Shapiro, J. Clin. Invst. Ag: 321-328 (1981) with the following modifications: 1. The Neuraminidase used was from Vibrio cholera type II (Sigma).

2. The reaction mixture contained 0.2 Units enzyme/ mg protein and protease inhibitors according to the following concentrations: Benzamidine (20 mM), Leupeptin (15 Ag/ml) and Aprotinin 20 ml). The asialo-vWF was used for platelet aggregation without any further purification.

Platelet aggregation Induced bv Asialo-vWF As stated above, soluble vWF does not bind to platelets via the GPIb receptor. Asialo-vWF, obtained by neuraminidase treatment to remove sialic acid residues, readily binds to platelets via GPIb. Presumably, the desialation lowers the WO 91/13093 PCr/US91/016 -36net negative charge on the vWF, allowing it to bind to the negatively charged GPIb receptor. Asialo-vWF binding to platelets causes activation, release of ADP, and GP IIb/ IIIa mediated aggregation. Platelet aggregation induced by asialo-vWF was carried out with 200 1l of PRP (Platelet-rich plasma) (Fujimura et al., J. Biol. Chem. 2M1: 381-385 (1986)) and 39 Ag/ml of asialo-vWF (final concentration) in a Lumi aggregometer. The results of inhibition of platelet aggregation with VC, the vWF GPIb binding domain polypeptide, are summarized in Table I.

VC (also referred to as VCL or VC3) is a vWF GPIb binding domain polypeptide which includes methionine plus amino acids 504-728 (see Figure 12).

vWF-Ristocetin induced platelet aqgregation Ristocetin-induced platelet aggregation in the presence of purified human intact vWF was carried out with washed human platelets according to Fujimura Y. et al., J. Biol. Chem.

261: 381-385 (1986).

The results of inhibition of platelet aggregation induced by ristocetin in the presence of intact vWF are summarized in Table II. Additional results using these assays are described in Example WO 91/13093 PCr/US91/01416 -37- Inhibition of Asialo-vWF Induced Platelet Aggregation (In PEP) by VC, a vWF GPIb Binding Domain Polypeptide QSpaoeFa-VC IInhibition of~ tiepaoneFrc concentration Platelet Aggregationio MM ti 4r 200 mM NaCI 6 76 64 250 mM NaCi 6 82 73 500 nM MaCi 6 89 79 WO 91/13093 WO 9113093PCTIUS91 /01416 -38- Inhibition .by VC of Ristocetin Induced Platelet Aggregation a vWF GPIh Binding Domain Polypeptide Q-Sepharospe Frac- VC %'Inhibition of tion concentration in Platelet Aggre- MM gation 200 kt!A Nad 10 76 6 69 3 38 1. 22 250 mM NaCl 10 86 6 67 3 44 1 34 500 mM Nadl 10 100 6 79 3 68 2. 54 0.25 38 BDialysis Buffer 0 0 H(control) WO 91/13093 PC/US91/01416 -39xample 4: An Improved Method of Obtaining Pure. Oxidized. Folded and Biolotically Active vWF GPIb Binding Domain Polvyeptide In Example 2, fermentation of cells harboring plasmid pvWF- VC3 was ilescribed. Subsequently, a preferred plasmid, pvWF- VCL was onstucted as described in Example 1 and maintained in E.coli strain A4300. This host/plasmid system was fermented essentially as known in the art for vectors containing a gene expressed under control of the XPL promoter, see, for example coassigned EPO Patent Publication No. 173,280, published March 5, 1986, Example 5, pages 73-74 (without added biotin, thiamine, trace elements, and ampicillin). In this improved method of purification of vWF GPIb binding domain polypeptide, a cell pellet of the above fermentation of A4300/pvWF-VCL was used.

In this improved method a purer and more active polypeptide is produced than by the method disclosed in Example 2. The general scheme of the downstream process consists of steps A through H as follows: A. Cell disruption and suspension of pellet: A pellet containing the vWF GPIb binding domain polypeptide is obtained as described in Example 2, by sonication and centrifugation of a cell suspension in 50mM Tris-HCl pH=8, 50mM NaCl, imM EDTA, 1mM DTT, 1mM PMSF, and Glycerol, The pellet containing the inclusion bodies is dissolved at about 10% w/v in a solution such that the final concentrations after dissolution are 8M urea, 20mM DTT, HEPES pH 8, and 100mM NaC1. The resulting solution may be further purified by ion exchange chromatography as WO 91/13093 PCT/US91/01416 described below. Alternatively, the inclusion bodies may be solubiliznd in a buffer containing 6M guanidine hydrochloride iullowed by buffer exchange to urea. The inclusion bodies may also be dissolved at different concentrations of urea, guanidine hydrochloride or any other denaturant or in the absence of denaturants, for example, at extremes of pH.

B. Cation exchange chromatography: This step eliminates most of the contaminants and produces the vWF GPIb binding domain polypeptide at greater than 90% purity.

Any cation exchange carboxymethyl) method may be used in this step, but CM-SephLrose fast flow (Pharmacia) chromatography is preferred. The functional group may be carboxymethyl, a phospho group or sulphonic groups such as sulphopropyl. The matrix may be based on inorganic compounds, synthetic resins, polysaccharides, or organic polymers; possible matrices are agarose, cellulose, trisacryl, dextran, glass beads, oxirane acrylic beads, acrylamide, agarose/polyacrylamide copolymer (Ultrogel) or hydrophilic vinyl polymer (Fractogel). In a specific embodiment, the polypeptide is loaded onto a CM-Sepharose FF column equilibrated with SM urea, 1mM DTT, 20mM HEPES pH 8, 100mM NaCl. Pure polypeptide elutes in 8M urea, 1mM DTT, 20mM HEPES pH 8 and 200mM NaCl. Up to about

OD

280 units of solubilized inclusion bodies may be loaded per ml CM-Sepharose FF. At this ratio the eluted polypeptide typically has a concentration of 0D 28 0 /ml.

C. Oxidation/Refoldina: The polypeptide solution eluted from the cation exchange step above is treated with 6M guanidine hydrochloride (GuCl) to disrupt any aggregates.

The polypeptide is then diluted to 0.05 OD 280 /ml in 2M GuCl, pH 5-11, preferably 20mM HEPES pH 8, 0.1mM GSSG WO 91/13093 PCr/US91/01416 -41- (glutathione, oxidized form). This mixture is allowed to stand overnight at room temperature. The products are analyzed by gel filtration on fast protein liquid chromatography (FPLC) such as Superose 12 before proceeding. Analysis shows that this protein concentration reproducibly yields about 30% correctly oxidized monomers, and 70% S-linked dimers and multimers, as well as reduced and incorrectly oxidized monomers. A higher protein concentration gives a higher absolute yield of correctly oxidized monomers but a lower percentage yield due to increased formation of S-linked dimers and multimers. For example, a protein concentration of 0.1 OD 280 /ml yields only 20% correctly oxidized monomers. Reducing the concentration to 0.025

OD

280 /ml yields 35-40% correctly oxidized monomers but a lower absolute yield per liter oxidation. Oxidations may also be performed in urea instead of in GuCl, or in any other denaturant or in the absence of denaturants under appropriate buffer conditions in which, for example, pH, ionic strength, and hydrophobicity are varied. The preferred concentration of urea is in the range 0.5M to preferably 4M, and the preferred oxidant is GSSG in the range 0.01mM to 5mM preferably 0.1mM. Other oxidants such as CuCl 2 may be used or alternatively no oxidant may be added, thereby utilizing air oxidation only. For scale-up, 4M urea is the presently preferred solution for the oxidation step.

D. Concentration: The oxidation products are concentrated, preferably to about OD 280 =1 by a tangential flow ultrafiltration system with a 30K cutoff membrane, such as a "MINITAN" or "PELLICON" system of Millipore. The filtrate is quite clear as the material is relatively clean and most of the contaminants are large enough not to pass through the 30K membrane. It is thus possible to WO 91/13093 PC/US91/01416 -42reuse the filtrate for performing oxidations. This results in considerable savings since large volumes of 2M GuCl are quite expensive. No difference in the oxidation products of oxidations performed in reused versus freshly prepared 2M GuCl was detectable by FPLC analysis.

E. Dialysi: It is necessary to reduce the GuCl or urea concentration to less than 10mM. This is achieved by dialysis against 20mM HEPES pH8, lOOmM NaCl. The dialysis was performed in dialysis tubing with 2-3 changes of buffer, but may be alternatively performed by diafiltration against the same buffer in a tangential flow ultrafiltration system with a 10K MW cutoff membrane.

During dialysis, as the concentration of GuCl (or urea or other denaturant) decreases, a white precipitate forms.

This precipitate contains about 80% of the protein yielded by step D comprising S-S linked dimers, reduced and incorrectly oxidized monomer and some contaminants which coelutad from the cation exchange step. The supernatant is nearly 100% correctly oxidized and refolded monomer at a concentration of 0.2 OD 280 /ml, which is about 20% of the protein yield of step D. This selective precipitation of contaminants and undesirable forms of the protein as a result of dialysis was surprising and not predictable. The yield of correctly oxidized monomer can be greatly increased by recovery from the precipitate. This is done as follows: the solution is clarified by centrifugation. The supernatant is saved, and the pellet is treated with DTT to reduce S-S bonds and reoxidized as described above. The pellet is dissolved in a minimal volume of 6M GuCl, 20ml HEPES WO 91/13093 POW9US1/01416 -43pH 8, 150mM NaCl, 20mM DTT. The solution was passed through Sephadex G25 in a buffer similar to the dissolution buffer but containing only imM DTT (instead of 20mM). The eluate is then diluted to OD 280 -0.05 and treated as in steps C, D and E above. This procedure may be repeated more than once as long as additional purified monomer is obtained. All of the supernatants are then combined.

F. Cation exchanqe: The combined supernatant of the dialysate of step E is concentrated by binding to CM Sepharose in 20mM HEPES pH8, 100mM NaCI. Elution is with HEPES pH8, 400mM NaCl. The eluate is exclusively monomeric despite the high salt concentration.

Concentrations of up to 3 mg/ml have been achieved by this method and that is not the upper limit. This step can alternatively be performed with Heparin-Sepharose which also binds the purified monomer in 10mM Tris pH 7.4, 150mM NaCl. Elution from Heparin-Sepharose is performed using 10mM Tris-HCl pH 7.4, 500mM NaCl.

G. Dialjy§i: The product of the previous step is dialyzed against 20mM HEPES pH8, 150mM NaCl.

H. Storage: At this stage the purified vWF GPIb binding domain polypeptide may be lyophilized. Upon reconstitution in a volume of water equal to the volume before lyophilization, the resultant solution contains exclusively monomeric protein showing no traces of dimers or other multimers on FPLC.

In a specific embodiment of this method the following procedure was performed: WO 91/13093 P~/US91/01416 -44a) 10 gm inclusion bodies (comprising 0.43 g net dry weight) were dissolved in a final volume of 100ml 8M urea, DTT, 20mM HEPES pH 8, 100 mM WaC1.

b) The protein was loaded onto a CM Sepharose column equilibrated with 8M urea, imM DTT, 20mM HEPES pH 8, 100mM NaCl. The protein eluted at 200mM NaCL in 8M urea, HEPES pH 8, imM DTT, and was naved.

c) The saved eluate of the previous step was treated with 6M GuCl to eliminate any aggregates, and was then diluted to 0.05 OD 280 /ml in 2M GuCl, 20mM HEPES pH 8, 0.1mM GSSG.

Oxidation was performed overnight at room temperature.

(Note that the oxidation step can be performed in the presence of urea instead of GuCl') d) The oxidation products were concentrated to OD 280 =1 by ultrafiltration on a "MINITAN" unit containing a membrane.

e) The concentrate of the previous step was dialyzed with three buffer changes against 20mM HEPES pH 8, 100mM NaCl.

During dialysis, as the GuCl concentration decreased, a white precipitate formed which was removed by centrifugation and reprocessed once as described above. The supernatants were combined.

f) The combined supernatants were concentrated by binding to CM Sephyrose in 20mM HEPES pH 8, 100mM NaCl. The polypeptide was eluted in 20mM HEPES, pH 8, 400mM NaCl and stored at 4 0

C.

g) The saved eluate from the previous step was dialyzed WO V091/13093 PCNrUS9/01416 against 20mM HEPES pH 8, 150mM NaCl at 4 0

C.

h) After dialysis, the purified vWF GPIb binding domain polypeptide, designated VCL, was lyophilized.

Analysis of VCL 1. Amino acid sequence analysis of VCL purified as described above revealed that the N-terminal sequence is Met-Leu-His-Asp-Phe which is the expected sequence according to Figure 12 with the addition of an N-terminal methionine residue.

2. Examination of VCL on pol.yacrylamide gels revealed that VCL electrophoreses at lower apparent molecular weight under non-reducing conditions than under reducing conditions (beta-mercaptoethanol). This shift from compact to less compact configuration is consistent with the reduction of a disulfide bond. Such an intramolecular bond is formed between the cysteines at positions 509 and 695. (The shift in molecular weight is not large enough to be consistent with the redaction of an interr-7lecular bond.) WO 91/13093 P~/US91/01416 -46- Example Biolocical Activity of VCL. a vWF GPIb bindin domain The vwF GPIb binding domain polypeptide produced as described in Example 4 was designated VCL and was assayed for biological activity as described below.

1. Ristocetin induced platelet aigregation (RIPA RIPA assay was performed as described in Example 3 in a reaction mix containing 2x10 8 platelets/ml, lMg/ml plasma vWF, and 1mg/ml ristocetin. A series of concentrations of VCL was tested and the IC 50 of VCL in 3 assays was determined to be 0.2-0.3AM. 100% inhibition was achieved with about lpM VCL.

2. Asialo vWF induced Dlatelet aqcregation Asialo vWF induced platelet aggregation assay was performed as described in Example 3 with 200 1 p platelet-rich plasma (PRP) and 10g/ml asialo-vWF in a Lumi aggregometer. A series of concentrations of VCL was tested and the IC 50 of VCL in this assay was determined to be 0.15MM, and complete inhibition by 0.5 MM.

3. Effect of VCL on preformed areates The effect of VCL on preformed aggregates made by RIPA was tested. Aggregates were formed as in paragraph above in the absence of VCL. Addition of VCL to a concentration of disrupted the aggregates instantaneously.

WO 91/13093 PCT/S91/01416 -47- 4. Inhibition of thiombin induced Platelet aareation Thrombin induced platelet aggregation assay was performed using 0.025 unit/ml thrombin and stractan prepared platelets. A series of concentrations of VCL was tested and the IC 50 of VCL in this assay was determined to be 0.3MM.

This is a surprising effect, since in a parallel experiment, VCL was not effective in inhibiting direct binding of [1251] labelled thrombin to platelets.

Effect on platelet deposition under conditions of flow In a model system consisting of an everted denuded human umbilical artery in a flow cell, platelet deposition may be determined. Whole human blood flows over the artery fragment. After 10-15 minutes, the flow is stopped and platelet deposition is determined microscopically. The ICso of VCL in this system was determined to be about luM.

All the above results are summarized in Table III.

The inhibitory activity of VCL on ristocetin-induced or asialo vWF-induced platelet aggregation, ristocetin-induced vWF binding, and platelet adhesion was lost upon reduction of the disulfide bond between the cysteines at positions 509 and 695. In some experiments, the reduced VCL precipitated out of solution.

WO 91/13093 W091/3093PCI/US9i /01416 -48- TABL III Biological Activity of VCLI a vWF GPIb Binding Domain Polypeptide.

Example 5 Assay AM VCL (Paragraph No.) 1 Ristocetin induced 1C 50 =0.2-O.3 platelet aggregation 2 Asialo vWF induced 1C 5

O

0 platelet aggregation 3 Thrombin induced lC 50 =O.3 platelet aggregation 4 Dissolution of preformed aggregates Platelet deposition 1 5 under conditions of flow WO 91/13093 PCr/US91/01416 -49- Examale 6: Construction of Plasmid PVWF-VEL It was decided to construct a plasmid which expresses a slightly longer portion of the vWF GPIb binding domain than pvWF-VCL. The construction is shown in Figures 15-18 and described in the brief descriptions of the figures.

A. Construction of pvWF-VE2 Plasmid pvWF-VA2 (constructed as shown in Figure 4) was digested with NdeI and PstI and the large fragment isolated.

Four synthetic oligomers shown in Figure 16 were prepared.

Nos. 2 and 3 were treated with T4 polynucleotide kinase to add 5' phosphate. The above mentioned large fragment of pvWF-VA2 was ligated as shown in Figure 15 with the four oligomers (two kinased, and two non-kinased). The resulting plasmid shown in Figure 15 was designated pvWF-VE2.

B. Construction of plasmid pvWF-VE3 Plasmid pvWF-VE2 was digested with Ndel and HindIII and the 770 bp fragment containing the vWF GPIb binding domain was isolated. Plasmid pMLK-7891 was also digested with NdeI and HindIII and the large fragment was isolated. The resulting plasmid, shown in Figure 17, was designated pvWF-VE3.

C. Construction of Dlasmid PvWF-VEL Plasmid pvWF-VE3 was digested with XmnI, dephosphorylated with bacterial alkaline phosphatase (BAP) and then digested with NdeI and HindIII. Plasmid pMLK-100 was digested with NdeI and HindIII and dephosphorylated with BAP. The two digests were then ligated to yield plasmid pvWF-VEL as shown in Fi% rure 18. This plasmid expresses the DNA sequence WO 91/13093 PT/US91/01416 corresponding to amino acids 469-728 of mature vWF under the control of the APL promoter and the clI ribosomal binding site. The protein probably also includes an additional Nterminal methionine residue. A conservative base change was introduced into ala-473 changing GCC to GCA which also encodes alanine. This introduced an SphI site into the gene by changing GCCTGC to GCATGC.

Expression of pvWF-VEL in E.cli 4300(F-) yields a 29 kD protein which reacts strongly with a monocional anti-vWF antibody and will be referred to herein as VE WVO 91/131093 PC/US91/01416 -51- Exampi 7: Pharmaceutical Uses of vWF GPIb Binding Domain PolvDepdt.L Examples 1 and 4 describe the production and purification of a novel vWF GPIb binding domain polypeptide designated VCL.

Some of the uses envisaged for VCL or for other vWF GPIb binding domain polypeptides are described below.

Pharmaceutical compositions containing VCL or such other polypeptides may be formulated with a suitable pharmaceutically acceptable carrier using methods and carriers well known in the art.

1. The VCL composition described above may be used for prevention of platelet adhesion to damaged vascular surfaces T (see Example 5, sub-section 2. The VCL composition described above may be used for disruption of platelet-rich aggregates (see Example subsection 3).

3. The VCL composition described above may be used for prevention of re-occlusion following angioplasty or thrombolysis (see Bellinger et al., PNAS, USA, FA: 8100-8104 (1987), Prevention of occlusive coronary artery thrombosis by a murine monoclonal antibody to porcine von willebrand Factor).

4. The VCL composition described above may be used for prevention of platelet activation and thrombus formation due to high shear forces such as in stenosed or partially obstructed arteries or at arterial bifurcations (see Peterson et al., Blood 2: 625-628 (1987), Shear-induced platelet aggregation requires von Willebrand Factor and platelet membrane glycoproteins Ib and IIb-IIIa).

WO) 91/13093 PCT/US91/J1416 -52- The VCL composition described above may be ased for prevention of thrombosis and re-occlusion after angioplasty or thrombolysis due to thrombin activation of platelets (see Fuster et al., J. Am. Coll. Cardiol. 12: 78A-84A (1988), Antithrombotic therapy after myocardial reperfusion in acute myocardial infarction).

6. The VCL composition described above may be used for pr'vention of platelet adhesion to and aggregation on prosthetic materials (see Badimon at al., J. of Biomaterials Applications, 5: 27-48 (1990), Platelet interaction to prosthetic materials role of von Willebrand Factor in Platelet Interaction to PTFE).

7. The VCL composition described absve may be used for prevention of intramyocardial platelet 'igation in patients with unstable angina (see Davio et al., Circulation 23: 418-427 (1986), Intramyocardi?; platelet aggregation in patients with unstable angina suffering sudden ischemic cardiac death).

8. The VCL composition described above may be used for prevention of vasospasm and vasoconstriction following arterial injury caused by angioplasty, thrombolysis or other causes (see Lam et al., Circulation 2: 243-248 (1987), Is vasospasm related to platelet deposition?) 3. The VCL composition described above may be used for prevention of :c'enosis following angioplasty or thrombolysis (see '.,Bride et al., N. Eng. J. of Med. 21: 1734-1737 (198~3, Restenosis after successful coronary angioplasty).

The VCL composition described above may be used for prevention of vaso-occlusive crises in sickle-cell anemia WO 91/13093 IPC/US91/01416 -53- (see Wick et al., J. Clin. Invest. g0: 905-910 (1987), Unusually large von Willabrand multimers increase adnesion of sickle erythrocytes to human endothelial cells under controlled flow).

11. The VCL composition described above may be used for prevention of thrombosis associated with inflammatory response (see Esmon, Science 1348-1352 (1987), The regulation of natural anticoagulant pathways).

12. The VCL composition described above may be used for prevention of arteriosclerosis (see Fuster et al., Circulation Res. 51: 587-593 (1982), Arteriosclerosis in normal and von Willebrand pigs).

13. Phe VCL composi*ion described above may be used as an antimetastatic agent (see Kitagawa et al., Cancer Res.

49: 537-541 (1989), Involvement of platelet membrane glycoprotein Ib and IIb/IIIa complex in thrombin-depandent and -independent platelet aggregations induced by tumor cells).

WO 91/13093s PCI/US91/01416 -54- 1. In Vitro Studies For these studies VCL, or vehicle control, was made up fresh in sterile water (2.2 mg/ml stock).

A. Platelet Aagregation (PRP) This is a standardized von Willebrand Factor (vWF)-dependent aggregation assay using human or rat platelet rich plasma (PRP). The addition of various concentrations of unfractionated Bothrops jararaca venom (BJV), which includes botrocetin and an additional thrombin-like component, or ristocetin results in an aggregatory response in the absence of any additional agent. Using ristocetin (1.5 mg/ml) as the agonist 43 Ag/ml VCL abolished the aggregation of human PRP. Ristocetin up to 5.0 mg/ml did not cause measurable aggregation of rat PRP. Using this assay system with BJV as the agonist VCL at 83 -g/ml slightly inhibited the response of human PRP (Figure 19) but not rat PRP (Figure It is concluded that ristocetin is not a suitable agonist for inducing vWF dependent aggregation in the rat. Further, it is not possible to monitor the effects of VCL Sx yivo using BJV-induced aggregation of rat PRP. However, VCL does inhibit vWF-dependent aggregation in human PRP in vitro.

B. Platelet Thrombin Receptor Assay This assay measures the inhibition of thrombin-induced platelet pro-coagulant expression and is briefly described below. Human washed platelets are incubated in a buffer which contains CaCl 2 factor Xa, prothrombin, and human alpha-thrombin for 60 min at 28°C. At the end of this WO 91/13093 PCr/US91/01416 period an aliquot is transferred into a buffer containing S2238 and EDTA (to prevent any further thrombin generation).

The S2238 reaction is terminated after 15 minutes at room temperature with acetic acid and the absorbance at 405 nm read. The amount of S2238 cleavage directly due to the added human alpha-thrombin is. estimated by including a control which contains no prothrombin and this value is subtracted from all results. VCL was tested in this assay at a final concentration of 0.1 mg/ml.

This assay is sensitive to both thrombin inhibitors and thrombin receptor antagonists. In the presence 'f VCL the thrombin generation was 114% of control We therefore conclude that VCL is not a thrombin receptor antagonist in this system.

2. In Vivo ISt9Ses Arterial Thrombosis Model (Rat) This method is essentially a modification of the model of Shand et al., Thromb. Res. A 505-515 (1987). The method we use routinely is outlined below.

Rats are labelled with 111 1n platelets and 125I fibrinogen.

The dorsal aorta is clamped, using modified Spencer-Wells forceps, for 1 minute. After a 45 minute reperfusion period the damaged vessel is removed, washed in citrate and counted. Results are expressed as mg blood equivalents.

Differences in radiolabel accumulation between placebo and drug-treated animals are calculated and expressed as a percentage inhibition.

For the purpose of the evaluation of VCL the route of WO 91/13093 PCr/US1/01416 -56administration was by bolus intravenous injection. VCL was used at doses of 2mg/kg and 4mg/kg It was administered 1 minute prior to clamping. The vessel was then reperfused for twenty minutes. The antithrombotic effect was assessed at the 20 minute end point of the reperfusion. TLe shortening of the reperfusion time (as compared to routine) was designed to save compound.

Appropriate vehicle controls (n=5 for both doses) were assessed, It can be seen that under these conditions VCL inhibits thrombus formation in this mciel (Table IV). The inhibition is seen for the platelet (IIIIn) components of the thrombus at the 4mg/kg dose. The other changes do not reach statistical significance, thus VCL shows antithrombotic efficacy in this rat arterial model.

In conclusion, VCL exhibits an antithrombotic effect in this rat model of arterial thrombosis which may be dose dependent.

3. Discussion From the present data it appears that the VCL interacts with the human platelet vWF receptor and hence inhibits platelet aggregation in human PRP. There is however a marked difference between species (rat vs. human) when comparing inhibition of platelet aggregation. The species specificity of this effect and the causal mechanism were not investigated further. At a practical level this meant we were unable to analyze ex vivo samples in order to correlate the effects of VCL on aggregation and arterial thrombosis.

Hence the analysis and interpretation of the in vivo efficacy of VCL as an antithrombotic in the rat arterial thrombosis model is complicated by this factor.

WO 91/13093 PCT/US91/01416 -57- Despite GPlb possessing binding sites for both vWF and thrombin it would appear that any effects of VCL on thrombin binding to GPlb do not translate into antagonism of thrombin-induced pro-coagulant expression.

Overall VCL shows an antithrombotic effect in the rat arterial thrombosis model. This inhibition may be due to its interference with the binding of vWF to its receptor.

WO 91/13093 PCr/US91/1416 -58- The Effect of VCL on Arterial Thrombus Formation in the Rat Dorsal Aorta

INHIBITION

DOSE PLATELETS P FIBRINOGEN P N (mg/kg) 4 61.3 8.0 .01 34.7 8.7 NS 3 2 25.54 20.98 NS 22.78 13.48 NS The results are expressed as mean percentage inhibition standard error. The number of experiments in the treated groups; are denoted in the table and in all cases were compared to a group of 5 control animals. Statistical analysis was performed on the raw data prior to transformation to percentage inhibition. NS not statistically significant.

58a Microorganism Deposits: Accession No. Date of Deposit Depository ATCC 68530 ATCC 68241 15th February, 1991 26th February, 1990 ATCC 68242 ATCC 67703 26th February, 1990 23rd May, 1988 23rd May, 1988 American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, United States of America 921207,q:\oper\pas,74964-91.dep,58 ATCC 67704 ATCC 67705 ATCC 67706 23rd May, 1988 23rd May, 1988

Claims (31)

1. A non glycosylated, biologically active polypeptide having the amino acid sequence: X-A-[Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gin Lys Trp Val Arg Val Ala Val 'al Glu Tyr His Asp Gly Ser His Ala Tyr lie Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ile Ala Ser Gin Val Lys Tyr Ala Gly Ser Gln Val Ala Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gln lIle Phe Ser Lys lie Asp Arg Pro Glu Ala Ser Arg le Ala Leu Leu Leu Met Ala Ser Gln Glu Pro Gin Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gin Gly Leu Lys Lys Lys Lys Val lie Val Ile Pro Val Gly lie Gly Pro His Ala Asn Leu Lys Gin Ile Arg Leu lie Glu Lys Gin Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gin G1n Arg Asp Glu lie Val Ser Tyr Leu Cys]-B-COOH wherein X is N'12-methionine- or NH 2 A is a sequence of at least 1, but less than 35 amino acids, which sequence is present in naturally occurring human vWF, the carboxy terminal amino acid of which is the tyrosine #508 shown in Figure 12 herein; B is a sequence of at least 1, but less than 211 amino acids, which sequence is Spresent in naturally occurring human vWF, the amino terminal amino acid of which is the aspartic acid #696 shown in Figure 12 herein; and the two cysteines included within the bracketed sequence are joined by a disulfide bond.

2. A polypeptide of claim 1 having the amino acid sequence: '931101,p:\oper\jrw,74964bio.res,60 T~' -A: X-[Cys Ser Arg Leu Leu Asp Leu Val Phe Leu Leu Asp Gly Ser Ser Arg Leu Ser Glu Ala Glu Phe Glu Val Leu Lys Ala Phe Val Val Asp Met Met Glu Arg Leu Arg Ile Ser Gln Lys Trp Val Arg Val Ala Val Val Glu Ty His Asp Gly Ser His Ala Tyr Ile Gly Leu Lys Asp Arg Lys Arg Pro Ser Glu Leu Arg Arg Ie Ala Ser Gin Val Lys Tyr Ala Gly Ser Gin Val Ala Ser Thr Ser Glu Val Leu Lys Tyr Thr Leu Phe Gin le Phe Ser Lys Ile Asp Arg Pro Glu Ala Ser Arg le Ala Leu Leu Leu Met Ala Ser Gin Glu Pro Gin Arg Met Ser Arg Asn Phe Val Arg Tyr Val Gin Gly Leu Lys Lys Lys Lys Val le Val le Pro Val Gly Ile Gly Pro His Ala Asn Leu Lys Gin le Arg Leu le Glu Lys Gln Ala Pro Glu Asn Lys Ala Phe Val Leu Ser Ser Val Asp Glu Leu Glu Gin Gin Arg Asp Glu Ie Val Ser Tyr Leu Cys Asp Leu Ala Pro Glu Ala Pro Pro Pro Thr Leu Pro Pro Asp Met Ala Gin Val Thr Val Gly Pro Gly Leu Leu Gly Val Ser Thr Leu Gly Pro Lys]-COOH wherein X is NH 2 -methionine- or NH 2

3. A pharmaceutical composition comprising an amount of the polypeptide of claim 1 or claim 2 effective to inhibit platelet aggregation and a pharmaceutically acceptable carrier.

4. A method of inhibiting platelet aggregation which comprises contacting platelets with an amount of the polypeptide of claim 1 or claim 2 effective to inhibit platelet aggregation so as to inhibit platelet aggregation. An expression plasmid encoding the polypeptide of claim 1.

6. An expression plasmid encoding the polypeptide of claim 2 designated pvWF- VC3 and deposited under ATCC Accession No. 68241.

7. An expression plasmid encoding the polypeptide of claim 2 designated pvWF- VCL deposited under ATCC Accession No. 68242.

8. A bacterial cell which comprises the expression plasmid of claim 5, 6, or 7. 93 1025,p: \oper \ji74 964bio.res,61

9. An Escherichia coli cell of claim 8. A method of producing the polypeptide of claim 1 or claim 2 which comprises transforming a bacterial cell with an expression plasmid encoding the polypeptide, culturing the resulting bacterial cell so that the cell produces the polypeptide encoded by the ple ;mid, and recovering the polypeptide so produced.

11. A method of treating a subject with a cerebrovascular disorder which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to inhibit platelet aggregation.

12. A method of treating a subject wi'h a cardiovascular disorder which comprises administering to the subject an amount of the polypeptide of claim. 1 or claim 2 effective to inhibit platelet aggregation.

13. A method of treating a subject in accordance with claim 12, wherein the cardiovascular disorder comprises acute myocardial infarction.

14. A method of treating a subject in accordance with claim 12, wherein the cardiovascular disorder comprises angina. A method of inhibitirg platelet aggregation in a subject prior to, during, or after the subject has undergone angioplasty, thrombolytic treatment, or coronary bypass surgery which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to inhibit platelet aggregation.

16. A method of maintaining blood vessel patency in a subject prior to, during, or after the subject has undergone coronary bypass surgery, which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to inhibit platelet aggregation.

17. A method of treating a subject having cancer which comprises administering o~iniQ n-knrvP~ikim- 74Q4hin rpz 62 to the subject an amount of the polypeptide of claim 1 or claim 2 effective to retard tumor metastasis.

18. A method of inhibiting thrombosis in a subject which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to inhibit the thrombosis.

19. A polypeptide in accordance with claim 1 or claim 2 bound to a solid matrix.

20. A method of treating a subject suffering from platelet adhesion to damaged vascular surfaces which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to inhibit platelet adhesion to damaged vascular surfaces.

21. A method of preventing platelet adhesion to a prosthetic material or device in a subject which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to prevent platelet adhesion to the material or device.

22. A method of inhibiting re-occlusion in a subject following angioplasty or thrombolysis which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to inhibit re-occlusion.

23. A method of preventing vaso-occlusive crises in a suc~ect suffering from sickle cell anemia which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to prevent vaso-occlusive crises.

24. A method of preventing arteriosclerosis in a subject which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to prevent arteriosclerosis. A method of thrombolytic treatment of thrombi-containing, platelet-rich 931019,p:\oper\jmw,74964bio.res,63 /o3 aggregates in a subject which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to treat thrombi-containing, platelet- rich aggregates.

26. A method of preventing platelet activation and thrombus formation due to high shear forces in a subject suffering from stenosed or partially obstructed arteries which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to prevent platelet activation and thrombus formation.

27. A method of preventing thrombin-induced platelet activation in a subject which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to prevent thrombin-induced platelet activation.

28. A method of preventing stenosis as a result of smooth muscle proliferation following vascular injury in a subject which comprises administering to the subject an amount of the polypeptide of claim 1 or claim 2 effective to prevent stenosis.

29. A method of claim 18, wherein the thrombosis is associate with an inflammatory response. A method for recovering the purified, biologically active polypeptide of claim 1 which comprises: producing in a bacterial cell a first polypeptide having the amino acid sequence of the polypeptide but lacking the disulfide bond; disrupting the bacterial cell so as to produce a lysate containing the first polypeptide; treating the lysate so as to obtain inclusion bodies containing the first polypeptide; contacting the inclusion bodies from step so as to obtain the first polypeptide in soluble form; treating the resulting first polypeptide so as to form the biologically active polypeptide; 931019,p\oper\jm,74964bio.res,64 recovering the biologically active polypeptide so formed; and purifying the biologically active polypeptide so recovered.

31. A method of claim 30, wherein the treatment step comprises contacting the polypeptide with a thiol-containing compound and disulfide.

32. A method of claim 31, wherein the thiol-containing compound is glutathione, thioredoxin, p-mercaptoethanol or cysteine.

33. A method of claim 30, wherein the contacting step is effected in the presence of a denaturant.

34. urea. A method of claim 33, wherein the denaturant is guanidine hydrochloride or A method of claim 33, wherein the recovery of the polypeptide of step (f) comprises removing the denaturant by dialysis.

36. A method of claim 30, wherein in step the polypeptide is purified by cation exchange chromatography.

37. A method of claim 36, wherein the first polypeptide is purified by cation exchange chromatography after step DATED this 19th day of October, 1993. BIO-TECHNOLOGY GENERAL CORP. By Its Patent Attorneys DAVIES COLLISON CAVE r in:"- S; rC. IS 't 1 0/r 931019,p\oper\jmv.374964bi.res,65