Peptides corresponding to the Factor VIII binding domain of the von Willebrand factor

Abstract: Peptides which inhibit the binding of von Willebrand Factor to Factor VIII. Monoclonal antibodies capable of specifically binding to the region of von Willebrand Factor containing the Factor VIII binding domain. Improved methods of preparing Factor VIII.

Description: Background of the Invention This invention relates to peptides which inhibit the binding of von Willebrand factor (vWF) to Factor VIII (FVIII). vWF and FVIII both have important but different functions in the maintenance of hemostasis. vWF participates in platelet-vessel wall interactions at the site of vascular injury whereas FVIII accelerates the activation of Factor X by Factor IXa in the presence of platelets and calcium ions. vWF and FVIII circulate in plasma as a noncovalently linked complex thought to be held together by both electrostatic and hydrophobic forces. vWF is thought to stabilize FVIII in vitro and prolong its half-life in the circulation. Consequently, in the absence of endogeneous vWF the circulating half-life of FVIII is markedly reduced. Since FVIII participates in the intrinsic pathway of blood coagulation, agents capable of interfering with the interaction of FVIII and vWF would alter the FVIII level in plasma and in this manner serve as anti-thrombotic agents. The peptides of the present invention have the ability to act as anti-thrombotic agents by their prevention of the binding of vWF to FVIII. They also have the ability to stabilize FVIII in an in vitro environment in which FVIII is being produced. In a first aspect, the present invention provides a von Willebrand Factor polypeptide having a molecular weight determined by SDS-PAGE of 29 kDa and a sequence which is a sequential subset of the following sequence: wherein X is Thr or Ala, the N-terminal amino acid of the polypeptide lies in the range 3 Ser to 44 Gly, the C-terminal amino acid of said polypeptide lies in the range 244 Leu to 285 Asn and the polypeptide is able to inhibit binding of von Willebrand Factor to Factor VIII. In preferred embodiment, the polypeptide has an amino-terminal sequence beginning with amino-terminal amino acid residue 3 Ser and ending approximately with carboxy-terminal amino acid residue 244 Leu. In a further embodiment, the polypeptide has an amino-terminal sequence beginning with amino-terminal amino acid residue 24 Glu and ending approximately with carboxy-terminal amino acid residue 265 Ser. In another embodiment, the polypeptide has an amino-terminal sequence beginning with amino-terminal amino acid residue 44 Gly and ending approximately with carboxy-terminal amino acid residue 285 Asn. In a second aspect, the invention provides a von Willebrand Factor polypeptide having the amino acid sequence: which is able to inhibit binding of von Willebrand Factor to Factor VIII. In a still further aspect, the invention provides an improved method of preparing Factor VIII by the addition of a polypeptide in accordance with the first or second aspects of the invention to Factor VIII from a recombinant produced, plasma or commercial concentrate source, to form a polypeptide/Factor VIII complex, subjecting the complex to purification procedures; recovering the complex and separating Factor VIII from said polypeptide. In another aspect, the invention provides a further improved method of preparing Factor VIII comprising: absorbing Factor VIII from a recombinant produced, plasma or commercial concentrate source onto particles bound to a polypeptide in accordance with the first or second aspect of the invention; eluting the Factor VIII; absorbing the eluted Factor VIII in another adsorption to concentrate and further purify same; eluting the absorbed Factor VIII; and recovering highly purified and concentrated Factor VIII. In yet another aspect, the invention provides a method of preparing a polypeptide in accordance with the first or second aspect of the invention by a recombinant DNA or synthetic peptide technique. In a still further aspect, the invention provides an improved method for expressing recombinant DNA produced Factor VIII comprising: expressing a polypeptide in accordance with the first or second aspect of the invention by recombinant DNA techniques in the same cell in which Factor VIII is expressed by recombinant DNA techniques; allowing a complex to form between said peptide and Factor VIII either in the cell, during secretion from the cell, or after secretion from the cell; recovering the complex; and separating Factor VIII from said peptide. In another aspect, the invention provides a pharmaceutical composition for use in preventing thrombosis or in inhibiting binding of von Willebrand Factor to Factor VIII, comprising at least one polypeptide or peptide in accordance with the first or second aspects of the invention. Further preferred embodiments of the invention are set out in the sub-claims appended hereto. In contracting states GR and ES, the invention relates to the methods and uses recited in the set of claims appended hereto and identified as for effect in those states. A monoclonal anti-vWF antibody, C3, was found to have the ability to block the binding of purified human FVIII to purified human vWF in a crossed immunoelectrophoresis system. Thus, the epitope of C3 must reside close to that of the FVIII binding domain of vWF and this antibody was therefore used as a marker of the FVIII binding domain. Whole unreduced ¹²⁵I-labeled vWF was treated with subtilisin at a 1/25 (w/w) ratio for 24 hours at room temperature. This reaction mixture was then placed in microtiter wells which had previously been coated with monoclonal anti-vWF antibody C3. The wells were thoroughly washed and then treated with SDS buffer heated to approximately 90°C and the solution run on a 5-15% gradient SDS-PAGE gel. An autoradiograph of the SDS-PAGE gel demonstrated predominately a single band with a molecular weight of approximately 29 kDa. A similar digest of unlabeled vWF was made and this reaction mixture was placed on chromatography column made up of monoclonal anti-vWF antibody C3 coupled to Sepharose 4B. The C3 reactive fragments were then eluted with 3M NaSCN, dialyzed, and concentrated. A band reactive with C3 by immunoblotting techniques was identified. Amino acid sequencing of this band revealed that approximately 60% of the amino-termini began with amino acid residue number 44 of the mature vWF subunit, approximately 20% began with residue number 24 and approximately 10% began with residue number 3. The above described experiment localized the C3 epitope and indirectly the FVIII binding domain to the amino-terminal region of vWF. Since the molecular weight of the peptide so identified was approximately 29 kDa and its predominant amino-terminus was amino acid residue 44 of the mature subunit, then the carboxy-terminus should be approximately at amino acid residue 285 based on an average molecular weight per amino acid residue of approximately 120. Based on the published amino acid sequence of vWF in Titani et al., Biochemistry 25, 3174-3184 (1986) it is possible to synthesize peptides from the region beginning with residue 3 and ending with amino acid residue 285 which comprises the region of vWF containing the FVIII binding domain. In Titani et al. the sequence analysis identified both Ala, and Thr at a molar ratio of about 4:1 at residue 26. In contrast, the nucleotide sequence of the lambda HvWF1 clone predicted Thr at residue 26 according to Sadler et al., Proc. Natl. Acad. Sci. USA 82, 6394-6398 (1985). This discrepancy can be due to polymorphism in the protein or to an error in cDNA replication during the preparation of the DNA library. In view of this uncertainty at residue 26, the amino acid at residue 26 is identified by X which represents an undetermined amino acid. These peptides can interfere with FVIII-vWF interaction and thus serve as antithrombotic agents. Additional monoclonal antibodies to this region can be produced which will also interfere with FVIII-vWF interaction and thus can also serve as anti-thrombotic agents. Experimental procedures used in localizing the C3 epitope and indirectly the FVIII binding to the 29 kDa polypeptide fragment are explained in more detail below when these same procedures are used in localizing the C3 epitope and indirectly the FVIII binding to the 170 kDa polypeptide fragment. The purification of FVIII from commercial factor VIII concentrate (Armour Pharmaceutical, Kankakee, IL), by immunoadsorbent chromatography with monoclonal anti-vWF antibody is described in Fulcher et al., Proc. Natl. Acad. Sci. USA 79, 1648-1652 (1982). FVIII preparations obtained by this method and used in the following experiments had specific activities of 2900-3800 units/mg. Purified vWF was obtained from commercial factor VIII concentrate (Armour Pharmaceutical, Kankakee, IL), by immunoadsorbent chromatography with a monoclonal anti-vWF antibody bound to Sepharose as described in Fulcher et al. The bound vWF was eluted by 3M NaSCN as described in Fujimara et al., J. Biol. Chem. 261, 381-385 (1986) and concentrated and desalted with a tangential flow Minitan ultrafiltration system (Millipore, Bedford, MA), with a 100,000 molecular weight cut off membrane. The protein was further dialyzed extensively against 0.05 M Tris, 0.15 M NaCl, pH 7.35 (TBS). Mouse monoclonal anti-FVIII and anti-vWF antibodies were produced, purified, and characterized and described in Fulcher et al, and Fujimara et al. Radioiodination of monoclonal anti-FVIII and anti-vWF antibodies were done according to the method of Fraker and Speck, Biochem. Biophys. Res. Commun. 80, 849-857 (1978), to a specific activity of 3-10 x 10⁹ cpm/mg. SP fragment-III was obtained by limited proteolysis of vWF with Staphylococcus aureus V8 protease (Sigma, St. Louis, MO), and purified by the method of Girma et al., Biochemistry 25, 3156-3163 (1986), with modifications as described by Titani et al., Biochemistry 25, 3171-3184. All fragments were dialyzed against TBS pH 7.35 before testing. The reduction and alkylation of vWF was performed as has been previously described in Fujimara et al. Two dimensional crossed immunoelectrophoresis of vWF was performed as described in Zimmerman et al., Immunoassays: Clinical Laboratory Techniques for the 1980's, pp. 339-349, Alan R. Liss, Inc., New York (1980), with the following modifications. Agarose was poured in a 1.5 cm strip at the bottom of a 10.2 cm x 8.3 cm piece of Gelbond (FMC Corporation, Rockland, ME). Purified vWF or fragments of vWF, FVIII, and 125-l labeled monoclonal anti-FVIII antibody were mixed in the sample well and electrophoresed. A second gel containing 125-250 ul of rabbit serum containing polyclonal anti-vWF antibodies was then poured and the second dimension was electrophoresed at right angles to the first dimension. Autoradiographs were made of the gels and compared to Coomassie brilliant blue staining of the gels. Competitive inhibition assay of FVIII binding to solid phase vWF: 50 ug of whole unreduced vWF in 1 ml of 0.01 M PO₄, 0.15 M NaCl, 0.02% NaN₃, pH 7.3 (PBS), was incubated with three 1/4 inch in diameter polystyrene beads (Pierce Chemical Company, Rockford, IL) per 16 mm in diameter tissue culture well for 2 hours at room temperature. The solution was removed and the wells and the beads were then blocked with 1 ml of PBS containing 0.05% Tween-20 and 3% human serum albumin for 1 hour at room temperature. The wells and the beads were stored in the blocking solution at 4°C for 16 hours to 10 days before use. The wells and beads were then washed x 3 with PBS 0.05% Tween-20 and incubated for 1 1/2 hours at room temperature with 1.3 ug of purified FVIII and 0-100 ug of the competitive ligand in 1 ml of 0.05M imidazole, 0.15 M NaCl, 0.02% NaN₃, pH 7.0, 3mM Cacl. The beads then were washed x 5 with PBS 0.05% Tween-20 and incubated for 1 1/2 hour at room temperature with 1.5 x 10⁶ cpm of ¹²⁵I-monoclonal anti-FVIII antibody C2 (specific activity 3.8 x 10⁹ cpm/mg), in 1 ml of PBS 0.05% Tween-20 containing 0.5% bovine gamma globulin. After incubation, the wells and beads were washed with PBS 0.05% Tween-20 x 2. The beads were then transferred to clean wells and washed an additional four times and separately counted. Total cpm in the absence of competing ligands ranged from 1340-2520 cpm in different experiments Background counts were those obtained when ¹²⁵I-monoclonal anti-FVIII antibody C2 was incubated with the vWF coated beads in the absence of FVIII. These ranged from 60-200 cpm. Protein concentrations were determined by the method of Bradford, Anal. Biochem.72:248-254 (1976), using bovine serum albumin as a standard. Crossed immunoelectrophoresis demonstrated complex formation between purified vWF and purified FVIII. This was shown by co-precipitation of ¹²⁵I-labeled monoclonal anti-FVIII antibody with unlabeled vWF only when purified FVIII was included in the sample well. In order to localize the FVIII binding domain, similar experiments were performed with vWF fragments obtained by Staphylococcus aureus V8 protease digestion. Limited digestion of vWF with Staphylococcus aureus V8 protease has been reported to produce primarily a single cleavage in vWF yielding two major fragments. SP fragment II is a 110-kDa homodimer containing the carboxy-terminal portion of the vWF molecule (residues 1366-2050) and SP fragment III is a 170-kDa homodimer containing the amino-terminal portion of the vWF molecule. This 170-kDa polypeptide fragment has an amino-terminal sequence beginning with amino-terminal amino acid residue 1 Ser and a carboxy-terminal amino acid residue extending no further than amino acid residue 1365-Glu according to the amino acid sequence published in Titani et al., Biochemistry, 25, 3171-3184 (1986). These two fragments represent 100% of the molecular mass of the vWF subunit. Complex formation was demonstrated between FVIII and the amino-terminal SP fragment III but not with the carboxy-terminal SP fragment II. This indicates that the amino-terminal SP fragment III in its homodimer form maintains the capability of interaction with FVIII in a qualitatively similar way as that of whole vWF. The carboxy-terminal SP fragment II in its homodimer form does not demonstrate this FVIII binding capability. The monoclonal anti-vWF antibody C3 largely inhibited complex formation between FVIII and vWF when it was included in the sample well, whereas 80 other monoclonal anti-vWF antibodies (tested in pools of 5 each) were without effect. C3 also inhibited complex formation between FVIII and SP fragment III in this system. Direct reactivity of C3 with SP fragment III was shown by adding ¹²⁵I-labeled C3 to a sample well containing purified SP fragment III. Autoradiographs of the crossed immunoelectrophoresis gel showed co-precipitation of the radiolabeled antibody with SP fragment III. In a similar experiment, no co-precipitation with SP fragment II occurred. In order to better characterize FVIII binding to vWF, a competitive inhibition assay was developed. In this assay purified vWF or SP fragment III was adsorbed to the surface of polystyrene beads. The beads were then incubated with purified FVIII. Purified FVIII bound to both unreduced vWF and unreduced SP fragment III which had been immobilized on the surface of the polystyrene beads. This was demonstrated by the binding of ¹²⁵I-labeled monoclonal anti-FVIII antibody to polystyrene beads sequentially incubated with vWF and FVIII. Both the binding of FVIII to vWF and the binding of ¹²⁵I-labeled monoclonal anti-FVIII antibody to FVIII were specific in this system as demonstrated by the following experiments. First, the binding of FVIII was shown to be dependent on the presence of vWF adsorbed to the surface of the polystyrene beads. When the polystyrene beads were coated with human serum albumin and then incubated with FVIII, followed by ¹²⁵I-labeled monoclonal anti-FVIII antibody, the counts per minute measured were only 2% of that seen with FVIII binding to vWF coated polystyrene beads. Secondly, when vWF coated polystyrene beads were not incubated with FVIII, the bead associated counts per minute were only 1% of that seen when the FVIII incubation was included. The reversibility of the binding of FVIII to the immobilized vWF could also be demonstrated. Dissociation of FVIII from the vWF-FVIII complex has been shown to occur in the presence of 0.25 M Cacl₂ according to Cooper et al., J. Clin. Invest. 54, 1093-1094 (1974), 10-20 mM EDTA according to Tran et al., Thromb. Haemostas. 50, 547-551 (1983) or 1-1.5 M NaCl according to Weiss et al., Thromb. Diath. Haemorrh. 27, 212-219 (1972). In the polystyrene bead system, five washings of the polystyrene beads with an imidazole buffered saline containing 0.25 M Cacl₂ at 37°C produced 70 +/- 4% dissociation of FVIII from vWF. Similarly, five washings with an imidazole buffered saline containing 20 mM EDTA produced 66 +/- 5% dissociation and with an imidazole buffer containing 1.5 M NaCl produced 86 +/- 1% dissociation of FVIII from vWF. Five washings with the same imidazole buffered saline containing 3 mM Cacl₂ produced no FVIII dissociation from vWF adsorbed to the polystyrene beads. The specificity of the binding of fluid phase FVIII to vWF immobilized to the surface of the polystyrene beads was also shown by the ability of whole, unreduced vWF in fluid phase to completely inhibit this binding. Reduced and alkylated vWF had no inhibitory effect on FVIII binding. Reduced and alkylated vWF, and reduced and alkylated SP fragment III, were also unable to bind FVIII in the crossed immunoelectrophoresis system. These findings are consistent with the observation that under mild reducing conditions FVIII can be dissociated from vWF, see Blomback et al., Thromb. Res. 12, 1177-1194 (1978). SP fragment III demonstrated dose dependent inhibition of FVIII binding with 90% inhibition at a concentration of 50 ug/ml. SP fragment I, a product of Staphylococcus aureus V8 protease digestion of SP fragment III which contains the middle portion of the vWF molecule (residues 911-1365 as described in Titani et al., Biochemistry 25, 3171-3184 (1986)) produced only 15% inhibition at concentrations up to 100 ug/ml. These data localized a major FVIII binding domain to the amino-terminal portion of vWF. SP fragment II inhibited FVIII binding by 29% at a concentration of 50 ug/ml. Doubling the concentration produced no significant increase in inhibition. The complete 2050 amino acid sequence of vWF has been determined by protein sequence analysis, see Titani et al., Biochemistry 25, 3171-3184 (1986). With such information a nucleotide sequence can be inserted into the appropriate vector for expression of the 29 kDa and 170 kDa polypeptide fragments and sequential subsets of polypeptide fragments which inhibit binding of vWF to FVIII. For a description of recombinant DNA techniques for cloning vWF fragments, see Ginsburg et al., Science 228:1401-1406 (1985) and Sadler et al., Proc. Nat. Acad. Sci. USA 82, 6394-6398 (1985). Peptides at least three amino acid residues in length beginning from the amino-terminal region of the 29 kDa polypeptide fragment are synthesized as described by Houghton et al., Proc. Natl. Acad. Sci. 82:5135 (1985). In the well known procedure for solid-phase synthesis of a peptide, the desired peptide is assembled starting from an insoluble support such as benzhydryl amine or chloromethylated resin (derived from cross-linked polystyrene, and available from chemical supply houses). The amino acid at the carboxy-terminal end of the desired polypeptide, carrying protecting groups on the alpha-amino nitrogen and on any other reactive sites, is attached to the resin from solution using known peptide coupling techniques. The protecting group on the alpha-amino group is removed (leaving other protecting groups, if any, intact), and the next amino acid of the desired sequence (carrying suitable protecting groups) is attached, and so on. When the desired polypeptide has been completely built up, it is cleaved from the resin support, all protecting groups are removed, and the polypeptide is recovered. Examples of suitable protecting groups are: alpha-tert-butyloxycarbonyl for the alpha-amino-group; benzyl, 4-methoxybenzyl, or 4-methylbenzyl for the thiol group of cysteine, the beta-carboxylic acid group of aspartic acid, the gamma-carboxylic acid group of glutamic acid and the hydroxyl groups of serine, threonine, and tyrosine; benzyloxycarbonyl or a 2-chloro- or 3-, 4-dimethoxy-derivative thereof for the ring nitrogens of histidine and tryptophan and the epsilon-amino group of lysine; p-nitrophenyl for the amide nitrogens of asparagine and glutamine; and nitro or tosyl for the guanidine group of arginine. For purposes of this disclosure, accepted short-hand designations of the amino acids have been used. A complete listing is provided herein below: One and three-letter Amino Acid Abbreviations A Ala Alanine C Cys Cysteine D Asp Aspartic Acid E Glu Glutamic Acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Glu Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine B Asx Asp or Asn, not distinguished Z Glx Glu or Gln, not distinguished X X Undetermined or atypical amino acid One or more of the peptides of the present invention can be formulated into pharmaceutical preparations for therapeutic, diagnostic, or other uses. To prepare them for intraveneous administration, the compositions are dissolved in water containing physiologically compatible substances such as sodium chloride (e.g. 0.35 - 2.0 M), glycine, and the like and having a buffered pH compatible with physiological conditions. The amount to administer for the prevention of thrombosis will depend on the severity with which the patient is subject to thrombosis, but can be determined readily for any particular patient. The following example is given as illustrative of the present invention. The present invention is not restricted only to this example. Example 1 Preparation of monoclonal antibody, C3, from hybridoma cell line In the procedure for production of the hybridoma cell line producing monoclonal anti-vWF antibody C3 mice of strain BALB/c (Research Institute of Scripps Clinic) were immunized intraperitoneally with purified FVIII immunogen containing small amounts of vWF which co-purified with it as a contaminant. The FVIII was prepared as described in Fulcher et al., Proc. Natl. Acad. Sci. USA 79, 1648-1652 (1982). The mice were immunized intraperitoneally with 1 ug of immunogen in complete Freund's adjuvant. Seven days later the mice were immunized intraperitoneally with 10 ug of immunogen in incomplete Freund's adjuvant. Seven days after this second injection they were immunized intraperitoneally with 50 ug of immunogen in incomplete Freund's adjuvant. Eight days after this third injection they were immunized intraperitoneally with 100 ug of soluble immunogen. Spleens were removed three days later, and spleen cells were fused with P3x63-AG8.653 (mouse myeloma cell line). P3X653-AG8.653 was maintained (before fusion) at log phase growth in a medium of 90% Dulbecco's modified Eagle's medium (high glucose) and 10% Fetal bovine serum (FBS). The following recommended supplements were added to 475ml of the above medium: glutamine (100x) 5 ml, sodium pyruvate (100x) 5 ml, nonessential amino acids (100x) 5ml, Pen-strep-fungizone (100x) 5 ml and 8-azaguanine 6.6 x 10⁻³M (50x) 10 ml. Spleen and myeloma cells were washed thoroughly without FBS in Dulbecco's modified Eagle's medium before fusion. Cells were fused with 1 ml 40% PEG 1500 for 1 minute. Then cells were diluted 1:2 with growth medium for 1 minute. Cells were diluted further 1:5 with growth medium for 2 minutes. Next cells were spun 900 RPM for 10 minutes. The supernatant was removed, the cells were selected by suspension in HAT medium and placed in 96 well plates. The HAT medium contained 90% Dulbecco's modified Eagle's medium (high glucose), 10% FBS and the following recommended supplements added to 405 ml of the above two components: glutamine (100x) 5 ml, NCTC 109 50 ml, sodium pyruvate (100x) 5 ml, nonessential amino acids (100x) 5 ml, Pen-strep-fungizone (100x) 5ml, (hypoxanthine 10⁻²M + thymidine 1.6 x 10⁻³M) (100x) 5 ml, bovine insulin (20 I.U./ml)(100x) 5 ml, oxaloacetate (10⁻¹M) (100x) 5 ml and aminopterin (2x10⁻⁵M)(50x) 10 ml. For 4 weeks following selection the cells were maintained in growth medium - HT (selection medium minus aminopterin). Subcloning was accomplished by limiting dilution. Wells with growth are tested by ELISA assay. Test plates were coated with 100ng/well immunogen or human fibrinogen, or human fibronectin, or human vWF, each protein being a potential contaminant of the immunogen. 50 ul of culture supernatant were tested. Those wells containing cells whose supernatants were positive with a vWF were grown at 37°C in 10% CO₂. For ascites production the mice were primed with 0.5 ml pristine at least 4 days before cell injection. The cells were injected intraperitoneally (5x 10⁶/mouse) in 0.5 ml media with no FBS. The ascites were harvested when the mice bloated. The monoclonal anti-vWF antibody C3 contained in the mouse ascites is of the IgG-1 type. The following Protein A sepharose purification of monoclonal anti-vWF antibody C3 from mouse ascites is a modified procedure of that disclosed in Ey et al. Immunochemistry, 15, 429-436 (1978). The amounts used were for a column 1 cm x 15 cm which bound about 25-30 mg IgG-1, but which allowed separation of about 50 mg IgG-1 from non-IgG proteins. The column can also bind 50 mg of IgG2a. IgG2b also binds to the column, but IgM, IgA and IgE do not bind. 4-6 ml of ascites was centrifuged at 30,000 rpm for 45 minutes. The lipids were removed on top. The addition of 20% sucrose weight/column to the ascites aided in the removal of lipids. Ascites was diluted to 25-30 ml with 140 mM NaPO₄ buffer, pH8, containing 0.02% NaN₃. The ascites was diluted to prevent the interference of chloride ion with the binding of IgG. Approximately 2 g of Protein A sepharose (Sigma) was swollen in 10 mM phosphate buffered saline with 0.02% NaN₃ and packed into a 1 cm diameter column. The column was equilibrated in 140 mM NaPO₄ buffer with 0.02% NaN₃. The column was loaded with diluted ascites at 0.06-0.08 ml/min or less. The column was allowed to sit at 4°C overnight after loading to increase binding of IgG. The column was washed with buffers at 0.6-0.8 ml/min in the following order: 1) 140mM NaPO₄ pH 8.0; 2) 0.1M Na citrate - citric acid, pH 6.0 (IgG-1 eluted); 3) 0.1 M Na citrate-citric acid, pH 5.0 - IgG2a eluted and a small percentage of remaining IgG-1; 4) 0.1 M Na citrate-citric acid (small percentage of remaining IgG2a eluted); and 5) 0.1M Na citrate-citric acid, pH 3.0 (IgG2b eluted). As soon as the column was washed with pH 3.0 buffer, it was washed with 140 mM NaPO₄ buffer, pH 8.0 + 0.02% NaN₃ until pH of effluent is 8.0. The column was stored at 4°C. During the washing of the column approximately 5 ml fractions were collected. To any fraction of pH 5.0, 1 ml of 1M tris HC1 was added.

Claims: Claims for the following Contracting States: AT, BE, CH, DE, FR, GB, IT, LI, LU, NL, SE A von Willebrand Factor polypeptide having a molecular weight as determined by SDS-PAGE of 29kDa and a sequence which is a sequential subset of the following sequence: wherein X is Thr or Ala, the N-terminal amino acid of the polypeptide lies in the range 3 Ser to 44 Gly, the C-terminal amino acid of the polypeptide lies in the range of 244 Leu to 285 Asn, and the polypeptide is able to inhibit binding of von Willebrand Factor to Factor VIII. A polypeptide as claimed in Claim 1 wherein said polypeptide has an amino-terminal sequence beginning with amino-terminal amino acid residue 3 Ser and ending approximately with carboxy-terminal amino acid residue 244 Leu. A polypeptide as claimed in Claim 1 wherein said polypeptide has an amino-terminal sequence beginning with amino-terminal amino acid residue 24 Glu and ending approximately with carboxy-terminal amino acid residue 265 Ser. A polypeptide as claimed in Claim 1 wherein said polypeptide has an amino-terminal sequence beginning with amino-terminal acid residue 44 Gly and ending approximately with carboxy-terminal amino acid residue 285 Asn. A von Willebrand Factor polypeptide having the amino acid sequence: which is able to inhibit binding of von Willebrand Factor to Factor VIII. An improved method of preparing Factor VIII comprising: (a) adding a polypeptide as claimed in any of claims 1 to 5 to Factor VIII from a recombinant produced, plasma or commercial concentrate source to form a polypeptide/Factor VIII complex; (b) subjecting the complex to purification procedures; (c) recovering the highly purified complex; and (d) separating Factor VIII from said polypeptide. An improved method of preparing Factor VIII comprising: (a) absorbing Factor VIII from a recombinant produced, plasma or commercial concentrate source onto particles bound to a polypeptide as claimed in any of claims 1-5; (b) eluting the Factor VIII; (c) absorbing the Factor VIII obtained in step (b) in another adsorption to concentrate and further purify same; (d) eluting the absorbed Factor VIII; and (e) recovering highly purified and concentrated Factor VIII. A method of preparing a polypeptide as claimed in any of claims 1-5 by recombinant DNA or synthetic peptide techniques. An improved method for expressing recombinant DNA produced Factor VIII comprising: (a) expressing a polypeptide as claimed in any of claims 1-5 by recombinant DNA techniques in the same cell in which Factor VIII is expressed by recombinant DNA techniques; (b) allowing a complex to form between said peptide and Factor VIII either in the cell, during secretion from the cell, or after secretion from the cell; (c) recovering the complex; and (d) separating Factor VIII from said peptide. A pharmaceutical composition for use in preventing thrombosis or inhibiting binding of von Willebrand Factor to Factor VIII, comprising at least one polypeptide as claimed in any of claims 1-5. Use of a composition comprising at least one polypeptide as claimed in any of claims 1-5 for the manufacture of a medicament for use in a method of preventing thrombosis. Use of a composition comprising at least one polypeptide as claimed in any of claims 1-5 for the manufacture of a medicament for use in a method of inhibiting binding of von Willebrand Factor to Factor VIII. Claims for the following Contracting States: ES, GR A method of preparing a von Willebrand Factor polypeptide having a molecular weight determined by SDS-PAGE of 29 kDa and a sequence which is a sequential subset of the following sequence: wherein X is Thr or Ala, the N-terminal amino acid of the polypeptide lies in the range 3 Ser to 44 Gly, the C-terminal amino acid of the polypeptide lies in the range of 244 Leu to 285 Asn, and the polypeptide is capable of inhibiting binding of von Willebrand Factor to Factor VIII, wherein said polypeptide is derived from a serum, or a recombinant DNA or synthetic peptide technique. A method of preparing a polypeptide as claimed in Claim 1 wherein said polypeptide has an amino-terminal sequence beginning with amino-terminal amino acid residue 3 Ser and ending approximately with carboxy-terminal amino acid residue 244 Leu. A method of preparing a polypeptide as claimed in Claim 1 wherein said polypeptide has an amino-terminal sequence beginning with amino-terminal amino acid residue 24 Glu and ending approximately with carboxy-terminal amino acid residue 265 Ser. A method of preparing a polypeptide as claimed in Claim 1 wherein said polypeptide has an amino-terminal sequence beginning with amino-terminal acid residue 44 Gly and ending approximately with carboxy-terminal amino acid residue 285 Asn. A method of preparing a von Willebrand Factor polypeptide having the amino acid sequence: wherein said polypeptide is able to inhibit binding of von Willebrand Factor to Factor VIII and is derived from a serum, or a recombinant DNA or synthetic peptide technique. An improved method of preparing Factor VIII comprising: (a) adding a polypeptide as recited in any of claims 1 to 5 to Factor VIII from a recombinant produced, plasma or commercial concentrate source to form a polypeptide/Factor VIII complex; (b) subjecting the complex to purification procedures; (c) recovering the highly purified complex; and (d) separating Factor VIII from said polypeptide. An improved method of preparing Factor VIII comprising: (a) absorbing Factor VIII from a recombinant produced, plasma or commercial concentrate source onto particles bound to a polypeptide as recited in any of claims 1-5; (b) eluting the Factor VIII; (c) absorbing the Factor VIII obtained in step (b) in another adsorption to concentrate and further purify same; (d) eluting the absorbed Factor VIII; and (e) recovering highly purified and concentrated Factor VIII. A method as claimed in any of claims 1-5 wherein the polypeptide is prepared by recombinant DNA or synthetic peptide techniques. An improved method for expressing recombinant DNA produced Factor VIII comprising: (a) expressing a polypeptide as recited in any of claims 1-5 by recombinant DNA techniques in the same cell in which Factor VIII is expressed by recombinant DNA techniques; (b) allowing a complex to form between said peptide and Factor VIII either in the cell, during secretion from the cell, or after secretion from the cell; (c) recovering the complex; and (d) separating Factor VIII from said peptide. A method of preparing a pharmaceutical composition for use in preventing thrombosis or inhibiting binding of von Willebrand Factor to Factor VIII, comprising taking at least one polypeptide or peptide as recited in any of claims 1-5, and compounding it with a pharmaceutical excipient. Use of a composition comprising at least one polypeptide or peptide as recited in any of claims 1-5 for the manufacture of a medicament for use in a method of preventing thrombosis. Use of a composition comprising at least one polypeptide or peptide as recited in any of claims 1-5 for the manufacture of a medicament for use in a method of inhibiting binding of von Willebrand Factor to Factor VIII.

Inventors: FOSTER PAUL A, FULCHER CAROL A, ZIMMERMAN THEODORE S, FOSTER, PAUL A., FULCHER, CAROL A., ZIMMERMAN, THEODORE S.
Applicants: SCRIPPS CLINIC RES, SCRIPPS CLINIC AND RESEARCH FOUNDATION
Application year: 1988
Main classification: C07K14
IPC codes: C12N15, A61K38, C07K14, C12P21, C07K16, C12N5, C07K1, A61P7, C12R1