WO1993016712A1 - FRAGMENTS DE GPIbα MUTANTS ET EXPRESSION RECOMBINEE DE CES FRAGMENTS - Google Patents

FRAGMENTS DE GPIbα MUTANTS ET EXPRESSION RECOMBINEE DE CES FRAGMENTS Download PDF

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WO1993016712A1
WO1993016712A1 PCT/US1993/001734 US9301734W WO9316712A1 WO 1993016712 A1 WO1993016712 A1 WO 1993016712A1 US 9301734 W US9301734 W US 9301734W WO 9316712 A1 WO9316712 A1 WO 9316712A1
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polypeptide
fragment
gpibα
glycoprotein ibα
sequence
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PCT/US1993/001734
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English (en)
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Zaverio M. Ruggeri
Jerry L. Ware
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The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to polypeptides which are useful in the treatment of vascular disorders such as thrombosis.
  • This invention further relates to the production by recombinant DNA-directed methods of pharmacologically useful quantities of the polypeptides of the present invention.
  • the term "hemostasis” refers to those processes which comprise the defense mechanisms of the body against loss of circulating blood caused by vascular injury. Processes which are normal as a physiologic response to vascular injury may lead in pathologic circumstances, such as in a patient afflicted with atherosclerotic vascular disease or chronic congestive heart failure, to the formation of undesired thrombi (clots) with resultant vascular occlusion. Impairment of blood flow to organs under such circumstances may lead to severe pathologic states, including myocardial - infarction, a leading cause of mortality in developed countries.
  • Primary hemostasis refers to the process of platelet plug or soft clot formation. Platelets are non- nucleated discoid structures approximately 2-5 microns in diameter derived from megakaryocytic cells. Effective primary hemostasis is accomplished by platelet adhesion, the interaction of platelets with the surface of damaged vascular endothelium on which are exposed underlying collagen fibers and/or other adhesive macromolecules such as proteoglycans and glycosaminoglycans to which platelets bind.
  • Therapeutic drugs for controlling thrombosis have been classified according to the stage of hemostasis which is affected by the administration thereof.
  • Such prior art compositions are typically classified as anticoagulants, thrombolytics and platelet inhibitors.
  • the anticoagulant therapeutics typically represent a class of drugs which intervene in secondary hemostasis. Anticoagulants typically have no direct effect on an established thrombus, nor do they reverse tissue damage. Associated with the use of existing anticoagulants is the hazard of hemorrhage, which may under some conditions be greater than the clinical benefits otherwise provided by the use thereof. As a result, anticoagulant therapy must be closely monitored. Certain anticoagulants act by inhibiting the synthesis of vitamin K-dependent coagulation factors resulting in the sequential depression of, for example, factors II, VII, IX, and X. Representative anticoagulants which are used clinically include coumarin, dicoumarol, phenindione, and phenprocou on.
  • Thrombolytics act by lysing thrombi after they have been formed.
  • Thrombolytics such as streptokinase and urokinase have been indicated for the management of acute myocardial infarctions and have been used successfully to remove intravascular clots if administered soon after thrombosis occurs.
  • the lysis effected thereby may be incomplete and nonspecific, i.e., useful plasma fibrinogen, in addition to fibrin polymers within clots, is affected.
  • a common adverse reaction associated with the use of such therapeutics is hemorrhage.
  • a third classification, antiplatelet drugs includes drugs which suppress primary hemostasis by altering platelets or altering or preventing their interaction with other circulatory system components.
  • the present invention relates to antiplatelet drugs.
  • a first example involves reducing the availability of ionized calcium within the platelet cytoplasm thereby impairing activation of the platelet and resultant platelet aggregation.
  • Pharmaceuticals representative of this strategy include prostacyclin, and also Persatine ®
  • An additional class of antiplatelet drugs acts by inhibiting the synthesis of thromboxane A 2 within the platelet, reducing the platelet activation response.
  • Non- steroidal anti-inflammatory agents such as ibuprofen, phenolbutazone and napthroxane may produce a similar effect by competitive inhibition of a particular cyclooxygenase enzyme, which catalyzes the synthesis of a precursor of thromboxane A 2 .
  • a similar therapeutic effect may be derived through the administration of aspirin which has been demonstrated to irreversably acetylate a cyclooxygenase enzyme necessary to generate thromboxane A 2 .
  • a third anti ⁇ platelet mechanism has involved the platelet membrane so as to interfere with surface receptor function.
  • One such drug is dextran, a large branched polysaccharide, which is believed to impair the interaction of fibrinogen with platelet receptors that are exposed during aggregation. Dextran is contraindicated for patients with a history of renal problems or with cardiac impairment.
  • the therapeutic ticlopidine is stated to inhibit platelet adhesion and aggregation by suppressing the binding of von Willebrand factor and/or fibrinogen to their respective receptors on the platelet surface.
  • ticlopidene possesses insufficient specificity to eliminate the necessity of administering large doses which, in turn, may be associated with clinical side effects.
  • the aforementioned pharmaceuticals are foreign to the body and may cause numerous adverse clinical side effects, there being no way to prevent such compounds from participating in other aspects of a patient's physiology or biochemistry, particularly if high doses are required. It would be desirable to provide for pharmaceuticals having such specificity for certain of the reactions of hemostasis, that they could be administered to patients at low doses, such doses being much less likely to produce adverse effects in patients.
  • EPO Publication No. 317278 discloses a method for inhibiting thrombosis in a patient by administering to the patient a therapeutic polypeptide comprised of the amino-terminal region of the chain of platelet membrane glycoprotein lb, or a.subfragment thereof (hereinafter the ⁇ chain is referred to as glycoprotein Ib ⁇ or "GPIb ⁇ .") .
  • the present invention is directed to the provision of modified (including mutant) antithrombotic polypeptides patterned upon glycoprotein Ib ⁇ .
  • a polypeptide patterned on a fragment of wild type glycoprotein Ib ⁇ having a predetermined affinity for von Willebrand factor said polypeptide having a modified sequence of amino acids relative to that of said fragment and an increased binding affinity, relative to said predetermined affinity, for von Willebrand factor ("vWF") .
  • the polypeptides of the invention exhibit a higher degree of therapeutic antithrombotic activity than the comparable wild type sequences.
  • the modified sequence of amino acids of a polypeptide of the invention may result from deletion of one or more amino acids (such as by mutation of an encoding DNA therefor) , or from covalent labeling of GPIb ⁇ polypeptide or of a fragment thereof.
  • a preferred modification results when, in comparison to a fragment of wild type GPIb ⁇ , one or more amino acid residues of said fragment are replaced with one or more amino acid residues found at the equivalent sequence positions of glycoprotein Ib ⁇ as isolated from one or more humans with platelet-type von Willebrand disease.
  • a preferred method for preparing polypeptides of the invention comprises the steps of:
  • A providing a DNA sequence encoding glycoprotein Ib ⁇ , or fragment thereof, in which one or more wild type codons thereof are replaced by codons specifying one or more amino acid mutations that confer upon the resultant expressed polypeptide enhanced binding affinity for von Willebrand factor relative to that of the comparable wild type sequence;
  • B inserting the DNA sequence so provided into a suitable plasmid or vector to create a construct comprising an expression plasmid or viral expression vector, said construct being capable of directing the expression of said polypeptide in a cell;
  • the process may be practiced with, for example, DNA sequences that encode GPIb ⁇ , or a fragment thereof, in which one or - more wild type codons thereof are replaced by codons specifying one or more amino acid mutations found in the glycoprotein Ib ⁇ DNA sequence of one or more patients having platelet-type von Willebrand disease.
  • an appropriate DNA sequence, encoding a GPIb ⁇ -derived polypeptide having increased binding affinity for vWF may be provided by random mutagenesis of the wild type DNA sequence using a randomized population of oligonucleotides.
  • a further aspect of the invention provides for a method of treating or inhibiting thrombosis in a patient which comprises administering to such patient an effective amount of one or more therapeutic compositions of the invention, said compositions comprising a pharmaceutically acceptable carrier and one or more polypeptides of the invention.
  • Figure 1 is a graph showing the ability of mutant and wild type GPIb ⁇ fragments to bind to vWF in the presence of ristocetin.
  • Cloning Vehicle - A plasmid, phage DNA or other DNA sequence which is able to replicate in a host cell, typically characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion for the insertion of heterologous DNA without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of expression control regions such as promoters or binding sites, and which may contain a selectable gene marker suitable for use in the identification of host cells transformed therewith, e.g., tetracycline resistance or ampicillin resistance.
  • a selectable gene marker suitable for use in the identification of host cells transformed therewith, e.g., tetracycline resistance or ampicillin resistance.
  • Plasmid - A nonchromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell.
  • the characteristics of that cell may be changed (or transformed) as a result of the DNA of the plasmid.
  • a plasmid carrying the gene for tetracycline resistance (Tet R ) transforms a cell previously sensitive to tetracycline into one which is resistant to it.
  • a cell transformed by a plasmid is called a "transformant.”
  • Expression Plasmid - A plasmid into which has been inserted the DNA being cloned, such as the glycoprotein Ib ⁇ structural gene.
  • the DNA sequence inserted therein may also contain sequences which control the translation of mRNA resultant therefrom, and contain restriction endonuclease sites which facilitated assembly of, and may facilitate further modification of, said expression plasmid.
  • An expression plasmid is capable of directing, in a host cell, the expression therein of the encoded polypeptide and usually contains a transcription promoter upstream from the DNA sequence of the encoded structural gene.
  • An expression plasmid may or may not become integrated into the host chromosomal DNA. For the purpose of this invention, an integrated plasmid is nonetheless referred to as an expression plasmid.
  • Viral Expression Vector - is similar to an expression plasmid except that the DNA may be packaged into a viral particle that can transfect cells through a natural biological process.
  • Table 1 shows the standard three letter designations for amino acids as used in the application.
  • the antithrombotic polypeptides of the present invention are based upon fragments of the natural occurring protein glycoprotein Ib ⁇ . For background purposes, there is set forth hereafter information concerning this protein and its role in hemostasis and thrombosis.
  • glycoprotein Ib-IX complex which consists of a noncovalent association of two integral membrane proteins, glycoprotein lb (GPIb) and glycoprotein IX (GPIX) .
  • GPIb which is a two-chain molecule having an apparent molecular mass of approximately 160 kDa, is composed of a heavy (alpha, or "GPIb ⁇ ") chain, having a molecular mass of approximately 145 kDa, linked by disulfide bonds to a light (beta, or GPIb/3) chain, having a molecular mass of approximately 22 kDa.
  • GPIb is an integral membrane protein and both the alpha- and beta- chains described above • have transmembrane domains.
  • the term "glycocalicin” refers to a soluble proteolytic fragment of the heavy ( ⁇ ) chain of GPIb that is generated by cleavage in a position close to the transmembrane domain of the molecule (Yamamoto, K. et al. Thromb. Res. , 43, 41-55 (1986)). It is now clear that glycoalicin comprises most of the extracellular domain of the GPIb ⁇ from which it derives.
  • the adhesive ligand of the GPIb-IX complex is the protein von Willebrand factor ("vWF") which is found as a component of the subendothelial matrix, as a component of the ⁇ -granules secreted by activated platelets, and also as a circulating blood plasma protein.
  • vWF von Willebrand factor
  • the actual binding site for vWF on the GPIb-IX receptor has been localized on the amino terminal region of the ⁇ chain of glycoprotein lb, specifically, to within a fragment thereof having a molecular weight of 45,000 (45 kDa) and comprising approximately a His'-Arg 293 fragment of GPIb ⁇ .
  • GPIb ⁇ and vWF perform essential roles in normal hemostasis during vascular injury and are also of central importance in the pathogenesis of acute thrombotic occlusions in diseased blood vessels. Both of these roles involve the interaction of vWF with platelets (at GPIb ⁇ ) which are induced to bind at the affected site, and are then crosslinked.
  • vWF glycoprotein Ib-IX complex
  • GPIb( ⁇ ) glycoprotein Ib-IX complex
  • the rapidly accumulating platelets are also crosslinked (aggregated) by the binding of fibrinogen at platelet glycoprotein Hb-IIIa receptor sites, and possibly also by vWF at these sites, and/or at additional glycoprotein Ib-IX receptor sites.
  • the glycoprotein lib/Ilia receptor may also be involved in the formation of the initial monolayer of platelets-
  • the multimeric and multivalent character of circulating vWF which enables the macromolecule to effectively carry out its binding and bridging functions, for example, to GPIb ⁇ .
  • these processes are normal as a physiologic response to vascular injury. However, they may lead in pathologic circumstances, such as in diseased vessels, to formation of undesired platelet thrombi with resultant vascular occlusion. Accordingly, inactivation or inhibition of the GPIb ⁇ receptors on the platelets of a patient would be of great medical importance for treating or inhibiting thrombosis. Accordingly, the present invention relates to the development of polypeptides which are effective in accomplishing the foregoing.
  • vWF contact by vWF with exposed or damaged subendothelium triggers its binding thereto and, possibly, vWF assumes an altered conformation (which is then capable of complex formation with GPIb ⁇ ) when ' contacting a blood vessel wall.
  • vWF assumes an altered conformation (which is then capable of complex formation with GPIb ⁇ ) when ' contacting a blood vessel wall.
  • Conformational changes necessary for this binding may also be induced in GPIb ⁇ by contact of the platelet with other blood components or exposure of the platelet to high sheer stress in a damaged vessel. Moake et al., Blood. 71, 1366-1374 (1988) .
  • vWF and GPIb ⁇ The interaction between vWF and GPIb ⁇ can be demonstrated in vitro by several methods. Binding can be demonstrated in the presence of ristocetin, a glycopeptide antibiotic which may act by reducing excess negative charge density between the macromolecules. See Howard et al.,
  • the gene for GPIb ⁇ has been cloned from a genomic cos id library utilizing a partial cDNA clone as a probe, and its sequence, including introns, has been determined by Wenger, R.H. et al. Biochemical and Biophysical Research Communications, 156(1), 389-395 (1988).
  • the GPIb ⁇ sequence predicted thereby consists of a 16 amino acid signal peptide. Met "16 through Pro "1 , followed by a 610 amino acid mature peptide or polypeptide region, His 1 through Leu 610 .
  • GPIb ⁇ The structure and properties of GPIb ⁇ are reviewed in Ruggeri, Z.M., The Platelet Glycoprotein Ib-IX Complex, in Progress in Hemostasis and Thrombosis, vol. 10, p.3568, Coller, B.S. ed. , W.B. Saunders Co., Philadelphia, 1991.
  • This invention provides antithrombotic polypeptides patterned on fragments of wild type glycoprotein Ib ⁇ polypeptide.
  • the polypeptides are effective in inhibiting activation and/or aggregation of platelets and adhesion of platelets to damaged or diseased vascular surfaces.
  • the invention reflects the discovery that polypeptides which have a modified sequence of amino acids relative to the fragments of wild type GPIb ⁇ upon which they are patterned and which possess increased binding affinity for vWF, relative to that of said wild type GPIb ⁇ fragments, are effective to treat or inhibit thrombosis. Without being limited as to theory, it is believed that such modified polypeptides are effective because they inhibit binding of vWF to platelet membrane- bound GPIb ⁇ . Additionally, modified polypeptides of the invention (mutant polypeptides, as described below) can be expressed from recombinant bacterial or eucaryotic host cells.
  • a polypeptide of the present invention comprises an amino acid sequence which is patterned upon the amino acid sequence of wild type glycoprotein Ib ⁇ , but an amino acid sequence which is structurally different in one or more respects than the amino acid sequence of said wild type glycoprotein Ib ⁇ , said difference in structure being such that the polypeptide of the present invention has an increased binding affinity for vWF.
  • a modified sequence of amino acids includes a difference in the sequence of amino acids of a fragment of glycoprotein Ib ⁇ polypeptide, compared to wild type sequence, without regard to how the difference is produced.
  • modifications with respect to one or more amino acid residues at one or more sequence positions in a polypeptide include deletions, additions, substitutions of amino acids, and also the covalent labelling of amino acids present therein, or the addition of amino acids containing such labels, i.e., radicals or blocking groups which affect the properties of the amino acid residues so labelled.
  • wild type amino acid sequence refers to the sequence of amino acids of a fragment of glycoprotein Ib ⁇ that is present in a large majority of humans. It is to be understood, however, that there are mutations in GPIb ⁇ , as isolated from certain persons, that do not affect significantly the interaction of GPIb ⁇ with vWF, for example, the Thr/Met 145 dimorphism. Fragments of GPIb ⁇ containing such mutations are also to be considered wild type.
  • glycoprotein Ib ⁇ fragments refer to GPIb ⁇ of humans
  • GPIb ⁇ , or a fragment of GPIb ⁇ , as described herein, whether "wild type” or “modified” refers to a polypeptide that may or may not contain one or more additional functional groups added by a mechanism of post- translational modification in a cell, such as glycosyl or sulfate groups.
  • additional functional groups may be added to polypeptide fragments of the invention by this mechanism or by an in vitro mechanism, including enzyme-catalyzed labelling or chemical labelling.
  • the invention encompasses biologically unimportant differences between the actual DNAs and polypeptides utilized in the practice of the invention and the structural sequences of amino acids or nucleotides thereof as reported herein.
  • the present invention provides modified fragments of GPIb ⁇ by, for example, mutation or covalent labelling so that the resultant polypeptides bind to vWF with a greater affinity than the comparable fragments upon which they are patterned.
  • GPIb ⁇ polypeptide fragments reflecting wild type GPIb ⁇ sequences can be used to inhibit binding of vWF to platelet-bound GPIb ⁇ in patients, it is desirable to identify polypeptides having increased affinity for vWF relative thereto which are therefore effective at lower doses. Provision of such polypeptides having an increased binding affinity allows for more effective clinical treatments including the provision of an effective dose by a lower concentration of therapeutic, with the result that potential adverse clinical consequences such as possible immune response are minimized.
  • a binding affinity is considered increased if the modification increases the therapeutic utility of the polypeptide by either: (1) increasing the affinity of the polypeptide for vWF relative to that of the comparable wild type fragment of GPIb ⁇ upon which it is patterned by at least about 10%, as measured in a suitable in vitro or in vivo assay; or (2) achieving an equivalent amount of binding to vWF with about 10% less polypeptide; or (3) with respect to a particular amino acid sequence modification, achieving (1) or (2) above by use of the said modification in combination with one or more other "modifications" to the amino acid sequence.
  • the antithrombotic therapeutics of the invention have an increased binding affinity of about 100% or more for vWF, and most preferably an increased binding affinity for vWF of about 5 fold or higher, compared to the wild type sequences of the GPIb ⁇ fragments upon which they are patterned.
  • Assays suitable for measuring "increased binding affinity" include (1) inhibition by GPIb ⁇ fragments of ristocetin-mediated binding of vWF to GPIb ⁇ on platelets; (2) inhibition by GPIb ⁇ fragments of botrocetin-mediated binding of vWF to GPIb ⁇ on platelets; (3) relative increase in ristocetin-mediated binding of vWF to immobilized GPIb ⁇ fragments (see Example 5 below) ; and (4) an increase in direct binding (without modulator) of vWF to immobilized
  • GPIb ⁇ fragments following generally the procedure in Example 5 below.
  • Trapani Lombardo, V. et al. , J. Clin. Invest.. 76, 1950-1958 (1985) provide procedures for immobilization of platelets useful in assays associated with techniques (1) and (2) above.
  • the following is a detailed procedure whereby, for example, 96 well polystyrene microtiter plates can be coated with formalin-treated platelets, said treatment not interfering with GPIb ⁇ function on the platelets.
  • the "comparable wild type sequence" is the His ⁇ Ala 302 fragment containing glycine (wild type residue) at position 233.
  • “Equivalent sequence position” means that the substituted amino acid (for example, Val 233 ) occupies the same position in the new polypeptide (position 233) as % in the amino acid sequence of GPIb ⁇ polypeptide from a patient having platelet-type von Willebrand disease from whom the mutation was identified, or that the substituted amino acid (for example, Val 233 ) occupies the same position in the new polypeptide that Gly 233 occupied in a wild type polypeptide — fragment.
  • the incorporation of a Gly ⁇ Val 233 mutation into a modified polypeptide would not affect the positions of Lys 231 , Gin 232 , Val 234 or Asp 235 .
  • An important aspect of the invention is the recognition that certain amino acid substitutions, additions or deletions- (compared to the wild type GPIb ⁇ sequence) can be made in fragments of GPIb ⁇ polypeptide to increase the binding affinity thereof for vWF, thereby making the resultant polypeptides more effective antithrombotics than the fragments of wild type GPIb ⁇ upon which they are patterned.
  • Polypeptides of the invention including such substitutions, additions and deletions can be made by any method known in the art, including chemical synthesis of a polymer of amino acids or, for example, from expression of an encoding DNA.
  • Increased binding affinity toward vWF can be similarly achieved by covalent labelling of certain wild type amino acid residues of GPIb ⁇ fragments.
  • the modified polypeptides of the invention are patterned on fragments of GPIb ⁇ that comprise all or part of the binding domain of GPIb ⁇ for vWF (the amino terminal residues 1-293 thereof) , and may contain also additional GPIb ⁇ polypeptide sequence.
  • the modified polypeptides of the invention represent fragments of GPIb ⁇ ranging in size from about 5 to about 500 amino acid residues.
  • the polypeptides of the invention do not contain the transmembrane domain of GPIb ⁇ , said domain commencing at approximately residue 486 of GPIb ⁇ polypeptide. are- hereafter described representative types and species of the modified polypeptides of the invention. There are described also methods to ascertain the identity of additional of such types and species.
  • Willebrand disease Such amino acid sequence mutations are considered to be responsible for said affliction, which is manifested by a prolonged bleeding time after injury or surgery.
  • Other manifestations of the disease, reflective of ' the underlying defect, are selective loss from circulation' of high molecular weight multimeric forms of vWF that are essential for hemostasis, and also mild thrombocytopenia.
  • vWD von Willebrand disease
  • vWD is type IIB, identified, for example, by an amino acid mutation Trp 550 ⁇ Cys 550 , located in the GPIb ⁇ binding domain of vWF. Ware, J. et al., Proc. Natl. Acad. Sci. USA. S8, 2946-2950 (1991).
  • Another subtype of vWD which is a relatively rare form of vWD, is termed either "pseudo" von Willebrand disease (Weiss, J. et al., N. Engl. J. Med.. 306, 326-333 (1982)), or "platelet-type” von Willebrand disease (Miller, J.L. and Castella, A., Blood, 60, 790-794 (1982), Miller, J.L. et al., J. Clin. Invest.. 72,
  • fragments of GPIb ⁇ that include one or more such mutations have, relative to wild type GPIb ⁇ fragments, increased binding affinity for vWF, and hence, increased utility as antithrombotics.
  • Example 4 provides an exemplary method whereby one or more mutations in GPIb ⁇ (such as Gly ⁇ Val) , indicative of platelet-type vWD, can be incorporated into GPIb ⁇ fragments providing thereby polypeptides which have improved activity, relative to fragments comprising comparable wild type GPIb ⁇ sequence, as antithrombotics.
  • Example 5 below demonstrates the improved antithrombotic utility (measured by increased affinity for vWF) of GPIb ⁇ fragment containing the Val 233 mutation.
  • Example 2 below provides an exemplary method for producing a His'-Ala 302 fragment of GPIb ⁇ that contains the vWF binding domain thereof.
  • Example 3 below demonstrates that this fragment, when expressed from recombinant mammalian cells (using pMW2 plasmid) possesses native tertiary conformation. Incorporation of the Val 233 mutation into the residue 1-302 fragment produces a polypeptide fragment having properties (Example 5) characteristic of full-length GPIb ⁇ in individuals afflicted with platelet-type vWD, for example, increased affinity for vWF at low doses of ristocetin.
  • glycoprotein Ib ⁇ that is associated with the platelet-type von Willebrand disease state is methionine to valine at GPIb ⁇ position 239.
  • antithrombotic polypeptides that comprise an amino acid sequence which corresponds to the amino acid sequence of fragments of GPIb ⁇ that include all, or a part of, the binding domain of GPIb ⁇ for vWF (approximately residues 1-293- thereof) , and which is modified in that the sequence includes one or more amino acids corresponding to those amino acid 5 residues of GPIb ⁇ responsible, in affected individuals, for platelet-type von Willebrand disease. It is expected that polypeptide fragments incorporating more than one such modification will, in many cases, be more effective as antithrombotics than polypeptide fragments comtaining only
  • This invention provides therefore for polypeptide fragments of GPIb ⁇ which incorporate one or more mutations associated with platelet-type vWD phenotype and have therefore, relative to the wild type sequence, an increased
  • the practice of the invention includes incorporation of Val 239 into GPIb ⁇ fragments to produce antithrombotic polypeptides.
  • the polypeptides demonstrate antithrombotic utility, that is, they prevent or
  • 35 mutations associated with platelet-type vWD can be incorporated into fragments of GPIb ⁇ , thereby providing increased binding affinity for vWF relative to comparable wild type fragments on which they are patterned even though said amino acid residue substitutions are not inserted into the polypeptide at the exact equivalent sequence positions that the residue mutations occupy in one or more GPIb ⁇ polypeptides from particular patients. It is understood that all such resulting polypeptides having similar therapeutic properties are within the scope of the invention.
  • the aforementioned antithrombotic polypeptides are made by a process of genetic engineering (recombinant DNA technology) which involves providing a DNA sequence that encodes a polypeptide containing one or more amino acid substitutions reflective of GPIb ⁇ as isolated from one or more patients with platelet-type vWD.
  • the types of amino acid substitutions useful in the design of said polypeptides include also use of amino acid mutations from presently unidentified patients, and/or from presently unsequenced or unidentified mutations of patients so afflicted.
  • Polypeptides of the invention may also be made by chemical synthesis (see, for example, Houghten, R.A. et al. , Proc. Natl. Acad. Sci. USA. 82, 5131-5135 (1985), or by enzymatic digestion of GPIb ⁇ or of a fragment thereof.
  • Gly 233 and Met 239 are semipolar amino acids, with methionine being capable, additionally, of hydrogen bond formation.
  • Reference to the published amino acid sequence of GPIb ⁇ (Titani, K. et al. , Proc. Natl. Acad. Sci. USA r 84, 5610-5614 (1987) shows that there are numerous other semipolar or hydrogen bonding-capable residues in the 220-250 subsequence of GPIb ⁇ located near residues 233 and 239, specifically Gin 221 , Asn 223 , Asn 226 , Tyr 228 , Trp 230 , Gin 232 , Thr 240 , Ser 241 , Asn 242 , Ser 245 , Gin 247 and Cys 248 .
  • the present invention includes within its scope a polypeptide which contains within its sequence, in place of one or more of the aforementioned residues, one or more bulky non-polar amino acid such as, for example, valine, leucine, isoleucine, tryptophan, and phenylalanine (capable of disrupting the functional roles of the aforementioned semipolar or hydrogen bonding-capable residues, thereby mimicking the likely effects of Val 233 and Val 239 in known platelet-type vWD cases) .
  • one or more bulky non-polar amino acid such as, for example, valine, leucine, isoleucine, tryptophan, and phenylalanine (capable of disrupting the functional roles of the aforementioned semipolar or hydrogen bonding-capable residues, thereby mimicking the likely effects of Val 233 and Val 239 in known platelet-type vWD cases) .
  • Such mutations define additional antithrombotic polypeptides.
  • polypeptide sequence by site-directed mutagenesis, after wild type Gin 232 and before Val 234 the following sequence designed to disrupt the structural role of Gly 233 : - Val 233' - Gly 233 - Val 233' .
  • residues such as Met 239 may be covalently labelled, such as by iodoacetamide to prevent the original side chain interactions (such as a hydrogen bond contact) of the wild type residue.
  • Serine and threonine residues may be labelled similarly by acetylation with acetyl chloride.
  • the practice of the invention includes also GPIb ⁇ polypeptide fragments that are expressed from recombinant procaryotic (bacterial) and recombinant eucaryotic cells (such as mammalian cells) .
  • recombinant procaryotic bacterial
  • recombinant eucaryotic cells such as mammalian cells
  • a major difference between fragments expressed from these two types of recombinant cells is the extent of posttranslational processing thereof.
  • polypeptides that are normally sulfated and glycosylated by recombinant host mammalian cells undergo substantially less of such ' modification if expressed in bacterial cells.
  • polypeptides of the invention include both glycosylated and nonglycosylated forms thereof, sulfated and non-sulfated forms thereof, and forms of GPIb ⁇ polypeptide fragment that were or were not subject to any other posttranslational modification characteristic of one or more types of host cell.
  • Glycosylation and sulfation may also be accomplished by enzymatic or chemical means in vitro.
  • _any gf_the _aboye strategies ⁇ (A) to (D.) can be used in any combination thereof sb ⁇ that, for example, one or more mutations from the GPIb ⁇ gene of one platelet-type vWD patient could be incorporated into a DNA encoding a therapeutic GPIbor'fragment along with one or more mutations from the DNA of another such patient, or along with one or more mutations not derived from a patient, but otherwise predicted or determined to confer antithrombotic properties. Similarly, more than one such predicted or determined mutation can be combined in an encoding DNA for expression therefrom of an antithrombotic polypeptide.
  • elements necessary for the practice of the invention are: (A) DNA sequences which encode a suitable fragment of GPIb ⁇ , such as the His'-Ala 302 encoding DNA sequence; (B) an expression plasmid or viral expression vector capable of directing in a cell the expression therein of the aforementioned fragment; and (C) a host bacterial or eucaryotic cell in which said expression may be effected.
  • the GPIb ⁇ polypeptide fragments so expressed are expected not to be secreted from host cells without attachment to the nascent GPIb ⁇ fragment of a signal peptide. Purification of proteins expressed therein and the extraction of pharmacologically useful quantities thereof is expected to be more difficult than if the GPIb ⁇ fragment could be caused to be secreted into the culture medium of the host cells.
  • Signal peptides corresponding to other protein species may prove equally effective to cause the secretion of GPIb ⁇ .
  • such fragments may be prepared to constitute, for example, one-fourth of a longer polypeptide expressed from a recombinant host cell, the longer polypeptide being next subject to proteolysis at a susceptible site to release the smaller polypeptide.
  • a wide variety of expression plasmids or viral expression vectors are suitable for the expression of the GPIb ⁇ polypeptide fragments.
  • One factor of importance in the selection of an expression system is the provision of a high efficiency transcription promoter directly adjacent to the
  • Another factor of importance in the selection of an expression plasmid or viral expression vector is the provision of an antibiotic resistance gene marker therein so that continuous selection for stable transformant eucaryotic host cells can be applied.
  • viral expression vector systems suitable for the practice of the invention including those based upon retroviruses and those based upon baculovirus Autographa californica nuclear polyhedrosis virus.
  • Representative host cells comprising permanent cell lines suitable for the practice of the invention include CHO- Kl Chinese hamster ovary cells, ATCC-CCL- 61; COS-1 cells, SV- 40 transformed African Green monkey kidney, ATCC-CRL 1650; ATT 20 murine pituitary cells; RIN-5F rat pancreatic ⁇ cells; cultured insect cells, Spodoptera frugiperda; or yeast (Sarcomyces) , and also strains of E. coli.
  • Examples 1 and 2 below contain an explanation of preferred procedures used to express the GPIb ⁇ polypeptide or an amino terminal fragment thereof. Details of these procedures are found in Murata, M. et al., J. Biol. Chem.. 266, 15474-15480 (1991) , and published International Application No. PCT/US91/00087 (published on July 11, 1991 bearing International Publication No. WO91/09614) .
  • Antibodies with Therapeutic Activity are powerful tools for analyzing the structure and function of macromolecules. By blocking macromolecular interactions, antibodies can also have important therapeutic utility. Accordingly, this invention includes within its scope an antibody which is specific for the GPIb ⁇ polypeptide, or any fragment thereof, and which is made by a process which involves immunizing animals with a polypeptide that itself is patterned upon a fragment of GPIb ⁇ and contains one or more amino acid residues corresponding to platelet-type von
  • compositions for therapeutic, diagnostic, or other uses.
  • the compositions are dissolved in water typically containing also one or more physiologically compatible substances such as sodium chloride; There results a solution having a pH, ionic strength, and osmotic potential compatible with therapeutic use (the range of potential solute concentrations being well known in the art, or readily determined) , said water and physiologically compatible substances comprise a pharmaceutically acceptable carrier.
  • the amount to administer for the prevention or inhibition of thrombosis will depend upon the affinity of the polypeptide for vWF in vivo f and/or for other macromolecules that participate in hemostasis and thrombosis in the body, on the lifetime of the polypeptide in the body, and on the severity with which the patient is subject to thrombosis. Said amount can be determined readily for any particular patient.
  • a therapeutic composition containing one or more of the polypeptides of the invention and also additional therapeutic substances.
  • additional substances include heparin and other anticoagulants, aspirin or other antiplatelet drugs, or tissue plasminogen activator or other prefibrinolytic drugs.
  • the signal peptide When attached at the amino terminal end of the residue 1-610 polypeptide (or residue 1- 302 fragment thereof, see Example 2) , the signal peptide causes the polypeptide to be recognized by cellular structures as a polypeptide of the kind to be processed for ultimate secretion from the cell, with concomitant cleavage of the signal peptide from the mature GPIb ⁇ polypeptide.
  • This example describes examplary conditions under which a DNA sequence encoding the fragment of mature GPIb ⁇ polypeptide having an amino terminus at His 1 and a carboxy terminus at residue Ala 302 thereof may be expressed, resulting in secretion from recombinant mammalian cells of the residue 1-302 GPIb ⁇ fragment.
  • This fragment contains sufficient primary sequence information to be assembled in a host cell into a polypeptide possessing domains of tertiary structure present in native glycoprotein Ib ⁇ and possessing the biological activity thereof. (see Example 3 below and Murata, M. et al., J. Biol. Chem.. 266, 15474-15480 (1991)).
  • the fragment corresponds to the 45 kDa amino terminal fragment (His ⁇ Arg 293 ) of GPIb ⁇ that contains the vWF binding domain thereof, and which can be produced by tryptic digestion of GPIb ⁇ . See Vicente, V. et al., J. Biol. Chem. , 263, 18473-18479 (1988), Vicente, V. et al., J. Biol. Chem.. 265, 274-280 (1990).
  • GPIb ⁇ antigen in stable transformant cells was demonstrated by applying cell lysates or culture medium from CH0-K1- containing dishes (both prepared as in Examples 9 and 10 of aforementioned International Application PCT/US91/00087) to nitrocellulose.
  • the 45 kDa GPIb ⁇ fragments (provided as respective culture media) were immobilized onto a nitrocellulose membrane (0.45 ⁇ pore size) placed at the interface between a 96-well sample application plate and a vacuum chamber. Culture medium from nontransformed CHO cells was used as control. Commercially available filtration apparatus (Pierce Chemical Co., Rockford, IL) and pump materials (Miniplus 2 , Gilson Co., Middleton, WI) were used. 30
  • Immobilization of the 45 kDa fragments was accomplished by causing a 200 ⁇ l volume of culture medium 5 (nonconcentrated) from the pMW2-transformed CHO cells to be vacuum-drawn through the nitrocellulose membrane over a 5 minute period. The protein binding capacity of the membrane was then saturated by passing through it three consecutive 200 ⁇ l aliquots of HEPES/BSA buffer, herein comprising ' 20 mM
  • ristocetin Sigma Chemical Co., St. Louis, MO
  • ristocetin Sigma Chemical Co., St. Louis, MO
  • Preincubation of the 50 ⁇ l aliquots was accomplished at room temperature for 30 minutes using various concentrations of ristocetin (0-2.0 g/ml) and a specified
  • the membrane was then allowed to dry and discs corresponding to the position of each application well were cut out and counted in a ⁇ scintillation spectrometer
  • Mutagenesis of a fragment (residues 1-302) of GPIb ⁇ to create additional GPIb ⁇ -derived polypeptides having properties reflective of platelet-type von Willebrand disease The following methods are representative of techniques which can be employed to (A) identify within the ' residue 1- 302 sequence of GPIb ⁇ additional amino acids of the wild type sequence thereof involved in modulating binding of GPIb ⁇ to vWF; and/or (B) to create artificial GPIb ⁇ -derived polypeptide sequences with enhanced binding activity toward vWF.
  • polypeptides that are soluble without such manipulations. Since formation of incorrect disulfide bonds (in or between individual polypeptides) is a difficulty associated with solubilizing proteins from inclusion bodies, it is preferred that random mutations be.expressed within relatively short polypeptides having few cysteine residues, and therefore a reduced tendency for disulfide bonding.
  • cysteine codons in the encoding DNA can be replaced by site- directed mutagenesis with inert codons such as those for serine or alanine.
  • such mutations may then be copied into a polypeptide comprising GPIb ⁇ amino acid sequence that is shorter or longer than the residue 221-318 subfragment, for example, the residue 1-302 fragment of GPIb ⁇ .
  • the resultant polypeptide may, if necessary, be expressed from a recombinant mammalian host cell, Example 2, to provide native conformation.
  • the appropriate size GPIb ⁇ DNA subsequence (for example, the encoding sequence for residues 221-318, Cruz, M. et al.) may be generated from pMWl or pMW2 DNA using, for example, the polymerase chain reaction. Random mutagenesis experiments can also be performed using GPIb ⁇ DNA constructs suitable for expression in mammalian cells such as those of Example 2, said expressed ' polypeptides appearing in stable soluble form in the culture medium.
  • Mutant oligonucleotides suitable for site directed mutagenesis protocols and spanning sequential 10 amino acid subdomains of the selected GPIb ⁇ sequence can be generated using a procedure designed to yield a randomly mutagenized oligonucleotide population.
  • the randomized GPIb ⁇ oligonucleotide is then hybridized, for example, to M13mpl8 bacteriophage containing an appropriate GPIb ⁇ -derived insert (for loopout mutagenesis) to copy the mutation into a residue 210-300 encoding DNA sequence.
  • the method of Hutchison, CA. et al. relies on automated synthesis of the oligonucleotide from the 3' end.
  • a random oligonucleotide population suitable for causing permutation of the residues between, for example, positions 230 and 250 of the mature GPIb ⁇ polypeptide would be constructed as follows.
  • the oligonucleotide corresponds to transcribed strand DNA.
  • each of the four nucleoside phosphoramidite reservoirs (A,T,G,C) for oligo- nucleotide synthesis would be "doped" with a small amount of each of the other three bases. Incorporation of one of the "doping" nucleotides would result in a mutant oligonucleotide. The amount of doping can be adjusted to control results.
  • the resultant randomized population of mutant oligonucleotides is then used in the standard site directed mutagenesis protocol (Example 4, see also Kunkel, T.A.
  • mutant clones can then be screened in GPIb ⁇ binding assays or in binding assays with GPIb ⁇ -specific monoclonal antibodies. Mutant clones having cell lysates which exhibit enhanced vWF binding or antibody response can be sequenced to determine the amino acid alteration(s) responsible for the
  • 35 mutant phenotype 35 mutant phenotype.
  • a very systematic analysis of the residue 210-300 region of vWF subunit can be performed and mutations which enhance the binding of GPIb ⁇ to vWF can be identified.
  • the process can be repeated, for example, for the residue 1-100, and 100-200 subfrag ents of the residue 1- 302 fragment of GPIb ⁇ .
  • the two known mutations which correlate with platelet- type von Willebrand disease result in replacement of a wild type codon, encoding a semipolar amino acid, with a codon corresponding to the nonpolar and bulky residue valine.
  • substitution for which is predicted to yield mutant polypeptides having platelet-type vWD properties and resultant antithrombotic therapeutic utility include Gin 221 , . Asn 223 , Asn 226 , ⁇ yr 228 , Trp 230 , Gin 232 , Thr 240 , Ser 241 , Asn 242 , Ser 245 , Gin 247 and Cys 248 .
  • Suitable replacements for the above amino acid residues include, as examples, isoleucine, valine, leucine, tryptophan and phenylalanine.
  • Cloned GPIb ⁇ polypeptide constructs reflecting known platelet-type vWD mutations may also be subject to the above mentioned random mutagenesis procedures and then screened for restoration of normal binding function such as, for example, having a normal response in a modulator-induced vWF binding assay. For example, it may be demonstrated that a particular "reversion" mutation proximal to residue 233 would compensate for, and nullify the effect of the original Gly ⁇ Val 233 mutation. The associated DNA sequence can then be determined to identify the relevant counteracting amino acid. Such procedures can be used to give important further evidence as to which other residue positions in the wild type amino acid sequence are important modulators of GPIb ⁇ binding.
  • a device used for the enzyme- linked immunofiltration assay technique Pierce Chemical Co., Rockford, IL, can be adapted in combination with immobilization of the mutant GPIb ⁇ -derived polypeptides to be tested. It is considered most efficient to initially test the effect of mutant codons on GPIb ⁇ polypeptides expressed from bacterial constructs and to then copy potentially useful mutations (using, for example, mutagenesis in M13mpl8 vehicle) into a mammalian expression construct. High levels of mutant GPIb ⁇ polypeptides corresponding to mutant DNA sequences can be expressed from, for example, pET- 3A type bacterial expression plasmids. Mutant polypeptides are expected to constitute a major portion of host E.coli cell lysates and can be readily screened for vWF affinity.
  • ELIFA enzyme- linked immunofiltration assay technique
  • site directed mutagenesis can be performed following the general procedure of Example 4 using as template in M13mpl8 the GPIb ⁇ -encoding sequence for the residue 210-300 subfragment of GPIb ⁇ .
  • oligonucleotide pool oligonucleotides each having, for example, randomly mutagenized residue 230 to 240 sequences are used.
  • the mutagenized population of M13mpl8 constructs can be cloned into pET-3A plasmids after which the expression plasmids can be transformed into host E.coli cells, for example, ampicillin sensitive strain BL21(DE3) , Novagen Co., Madison, WI.
  • Strain BL21(DE3) contains a gene for T7 RNA polymerase for high efficiency transcription. Preparation of mutant polypeptide extracts from E.coli BL21(DE3) for screening follows the procedure of Prior, C et al., if inclusion bodies develop. Otherwise, a procedure such as that of Cruz, M. et al. would be appropriate, or other procedures adaptable from the known art. Polypeptides corresponding to residues 1-100, 101-200, or 150-250 can be expressed similarly, and then tested as follows.
  • Resultant extracts of expressed mutant GPIb ⁇ polypeptide subfragments are immobilized following the manufacturer's instructions onto a nitrocellulose membrane (0.45 ⁇ pore size) using 96-well sample application plates (Easy-Titer ® ELIFA System, Pierce Co., Rockford, IL) and a vacuum chamber.
  • 96-well sample application plates Easy-Titer ® ELIFA System, Pierce Co., Rockford, IL
  • Commercially available pump materials can be used (see Example 5) .
  • the apparatus is suitable for screening large series of clone lysates in an ELIFA or dot blot system and allows also quantitative transfer of sample fluids to underlying microtiter wells without cross contamination.
  • Immobilization of the GPIb ⁇ polypeptides is accomplished by causing a suitable volume of GPIb ⁇ fragment solution, such as 200 ⁇ l, (including also 8 M urea if resuspended inclusion body pellet material is used) to be vacuum-drawn through the individual wells to the nitrocellulose membrane over a 5 minute period. Several 200 ⁇ l volumes of Hepes-buffered saline are then drawn through the membrane to remove urea.
  • a suitable volume of GPIb ⁇ fragment solution such as 200 ⁇ l, (including also 8 M urea if resuspended inclusion body pellet material is used)
  • the protein binding capacity of the membrane is then saturated by passing through it three consecutive 200 ⁇ l aliquots of HEPES/BSA buffer herein comprising 20 mM Hepes, pH 7.4, 150 mM NaCl, and 1% w/v bovine serum albumin (Calbiochem, La Jolla, CA) .
  • a 50 ⁇ l volume of HEPES/BSA containing 125 I-vWF (following the procedure of Example 5) which had been preincubated therein with ristocetin (at approximately 0-0.5 mg/ml) can be vacuum drawn through the nitrocellulose membrane again over a 5 minute period. See Example 5 above, for suitable preincubation conditions. Multiple plates may be used to screen at different ristocetin concentrations. The membrane is then allowed to dry and discs corresponding to the position of each application well are cut out and counted in a 7 scintillation spectrometer to determine bound radioactivity.
  • the counting process may be facilitated by scanning the developed autoradiogram in a densitometer to digitize the intensity of developed spots. As long as the autoradiogram is not excessively overdeveloped, beyond the linear region of response, useful qualitative results are obtained.
  • An alternate procedure to derive from individual host E.coli BL21 (DE3) clones an impure extract which can be screened in immunoblot or dotblot procedures is as follows. A large set of individual E.coli colonies carrying separate randomly mutagenized GPIb ⁇ residue 210-300 inserts is picked and grown overnight as separate cultures. The cultures are then diluted 1:100 and grown to an OD ⁇ of 1.0.
  • GPIb ⁇ fragment synthesis is induced by adding isopropyl-/3-d- thiogalactopyranoside (IPTG) , U.S. Biochemicals, Cleveland, OH, to 5 mM and continuing growth for approximately 2.5 hours.
  • IPTG isopropyl-/3-d- thiogalactopyranoside
  • the cells are harvested by centrifugation for 1 minute at 10,000 g and then washed and repelleted (at 10,000 g) 3 times with phosphate buffered saline (0.14 M NaCl, 0.1 M Na 2 HP0 4 pH 7.0).
  • the bacterial pellet is then solubilized by boiling for 10 minutes in a buffer comprising 0.01 M NaH 2 P0 4 , 10 mM Na 2 EDTA, 1% (w/v) sodium dodecylsulfate, pH 7.0.
  • GPIb ⁇ -derived polypeptides from colonies representing the most intense response are selected for confirmation (rescreening) of enhanced binding using methods such as those described above.
  • Clones which confer enhanced positive responses in these systems are then subjected to standard DNA sequencing procedures to identify the GPIb ⁇ gene mutations responsible for the mutant properties.
  • the appropriate mutations may be copied into a GPIb ⁇ DNA sequence within a plasmid suitable for expression in CH0-K1 cells, followed by further characterization therefrom.

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Abstract

Polypeptide calqué sur un fragment de la glycoprotéine Ibα de type sauvage présentant une affinité prédéterminée pour le facteur von Willebrand, ledit polypeptide présentant une séquence d'aminoacides modifiée par rapport à celle dudit fragment, ainsi qu'une affinité de liaison accrue pour le facteur von Willebrand, par exemple, par rapport à l'affinité prédéterminée. Ladite modification consiste à remplacer un ou plusieurs restes d'aminoacides dudit fragment par un ou plusieurs restes d'aminoacides se trouvant dans les positions de séquences équivalentes de la glycoprotéine Ibα isolée à partir d'au moins un homme souffrant de la maladie de von Willebrand de type plaquettaire. En outre, un procédé de production du polypeptide susmentionné à partir d'ADN codant la glycoprotéine Ibα ou un fragment de celle-ci, consiste, par exemple, à utiliser une séquence d'ADN dans laquelle un ou plusieurs codons de type sauvage sont remplacés par des codons spécifiant une ou plusieurs mutations d'aminoacide se trouvant dans la séquence d'ADN de la glycoprotéine Ibα d'au moins un patient souffrant de la maladie de von Willebrand de type plaquettaire. L'invention se rapporte également à une composition thérapeutique permettant de traiter ou d'inhiber efficacement les thromboses, et comprenant un excipient pharmaceutiquement acceptable et un polypeptide de l'invention, ainsi qu'à un procédé de traitement ou d'inhibition de la thrombose chez un patient, consistant à administrer à ce dernier une dose efficace de ladite composition thérapeutique.
PCT/US1993/001734 1992-02-26 1993-02-25 FRAGMENTS DE GPIbα MUTANTS ET EXPRESSION RECOMBINEE DE CES FRAGMENTS WO1993016712A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0701606A1 (fr) * 1993-04-23 1996-03-20 Bio-Technology General Corporation Procede d'amelioration de thrombolyse
US6251393B1 (en) 1998-10-23 2001-06-26 The Brigham And Women's Hospital, Inc. Conformation-specific anti-von Willebrand Factor antibodies
US6841354B2 (en) * 2000-12-21 2005-01-11 Philadelphia, Health & Education Corporation Screening assay for anti-thrombotic/anti-platelet activity
US7112661B1 (en) 1998-10-30 2006-09-26 The Research Foundation Of State University Of New York Variable heavy chain and variable light chain regions of antibodies to human platelet glycoprotein Ib alpha
WO2007077990A1 (fr) 2006-01-06 2007-07-12 Keio University Materiau de distribution de medicament
DE102007031708A1 (de) 2007-07-06 2009-01-08 Dade Behring Marburg Gmbh Bestimmung der von Willebrand Faktor-Aktivität in Abwesenheit von Ristocetin
US7608695B2 (en) * 1998-04-23 2009-10-27 Ajinomoto Co., Inc. Substance with antithrombotic activity and method for detecting glycokallidin

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0317278A2 (fr) * 1987-11-17 1989-05-24 Scripps Clinic And Research Foundation Fragments protéolytiques et peptides synthétiques qui bloquent le rattachement du facteur de von Willebrand à la glycoprotéine Ib de la membrane des plaquettes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0317278A2 (fr) * 1987-11-17 1989-05-24 Scripps Clinic And Research Foundation Fragments protéolytiques et peptides synthétiques qui bloquent le rattachement du facteur de von Willebrand à la glycoprotéine Ib de la membrane des plaquettes

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
BIOCHEMA ET BIOPHYSICA ACTA, Vol. 1097, issued 1991, PINCUS et al., "Conformational Energy Analysis of the Substitution of Val for Gly 233 in a Functional Region of Platelet GPIb-Alpha in Platelet-Type Von Willebrand Disease", pages 133-139. *
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 156, No. 1, issued 14 October 1988, WENGER et al., "Structure of the Human Blood Platelet Membrane Glycoprotein Ib-Alpha Gene", pages 389-395. *
BLOOD, Vol. 60, No. 3, issued September 1982, MILLER et al., "Platelet-Type Von Willebrand's Disease: Characterization of a New Bleeding Disorder", pages 790-794. *
BLOOD, Vol. 70, No. 4, issued October 1987, RUGGERI et al., "Von Willebrand Factor and Von Willebrand Disease", pages 895-904. *
J. BIOLOGICAL CHEMISTRY, Vol. 261, No. 27, issued 25 September 1986, HANDA et al., "The Von Willebrand Factor-Binding Domain of Platelet Membrane Glycoprotein Ib", pages 12579-12585. *
J. BIOLOGICAL CHEMISTRY, Vol. 265, No. 1, issued 05 January 1990, VICENTE et al., "Identification of a Site in the Alpha Chain of Platelet Glycoprotein Ib that Participates in Von Willebrand Factor Binding", pages 274-280. *
J. BIOLOGICAL CHEMISTRY, Volume 266, No. 25, issued August 1991, MURATA et al., "Site-Directed Mutagenesis of a Soluble Recombinant Fragment of Platelet Glycoprotein Ib-Alpha Demonstrating Negatively Charged Residues Involved in Von Willebrand Factor Binding", pages 15474-15480. *
J. BIOLOGICAL CHEMISTRY, Volume 267, No. 2, issued 15 January 1992, CRUZ et al., "Functional Analysis of a Recombinant Glycoprotein Ib-Alpha Polypeptide which Inhibits Von Willebrand Factor Binding to the Platelet Glycoprotein Ib-IX Complex and to Collagen", pages 1303-1309. *
J. CLIN. INVEST., Vol. 72, issued November 1983, MILLER et al., "Von Willebrand Factor Binds to Platelet and Induces Aggregation in Platelet-Type but not Type IIb Von Willebrand Disease", pages 1532-1542. *
J. CLIN. INVEST., Vol. 76, issued November 1985, TRAPANI et al., "Independent Modulation of Von Willebrand Factor and Fibrinogen Binding to the Platelet Membrane Gylcoprotein IIb/IIIa Complex as Demonstrated by Monoclonal Antibody", pages 1950-1958. *
METHODS IN ENZYMOLOGY, Vol. 154, issued 1987, KUNKEL et al., "Rapid and Efficient Site-Specific Mutagenesis without Phenotypic Selection", pages 367-383. *
PROC. NATL. ACAD. SCI. USA, Vol. 82, issued 15 August 1985, HOUGHTEN, "General Method for the Rapid Solid-Phase Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the Level of Individual Amino Acids", pages 5131-5135. *
PROC. NATL. ACAD. SCI. USA, Vol. 83, issued February 1986, HUTCHISON et al., "A Complete Library of Point Substitution Mutations in the Glucocorticoid Response Element of Mouse Mammary Tumor Virus", pages 710-714. *
PROC. NATL. ACAD. SCI. USA, Vol. 84, issued August 1987, TITANI et al., "Amino Acid Sequence of the Von Willebrand Factor-Binding Domain of Platelet Membrane Glycoprotein Ib", pages 5610-5614. *
PROC. NATL. ACAD. SCI. USA, Vol. 88, issued April 1991, WARE et al., "Identification of a Point Mutation in Type IIb Von Willebrand Disease Illustrating the Regulation of Von Willebrand Factor Affinity for the Platelet Membrane Glycoprotein Ib-IX Receptor", pages 2946-2950. *
PROC. NATL. ACAD. SCI. USA, Vol. 88, issued June 1991, MILLER et al., "Mutation in the Gene Encoding the Alpha Chain of Platelet Glycoprotein Ib in Platelet-Type Von Willebrand Disease", pages 4761-4765. *
SCIENCE, Vol. 252, issued 21 June 1991, WALDMANN, "Monoclonal Antibodies in Diagnosis and Therapy", pages 1657-1661. *
TIBTECH, Vol. 11, issued February 1993, HARRIS et al., "Therapeutic Antibodies - the Coming of Age", pages 42-44. *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0701606A4 (fr) * 1993-04-23 1999-06-30 Bio Technology General Corp Procede d'amelioration de thrombolyse
EP0701606A1 (fr) * 1993-04-23 1996-03-20 Bio-Technology General Corporation Procede d'amelioration de thrombolyse
US7608695B2 (en) * 1998-04-23 2009-10-27 Ajinomoto Co., Inc. Substance with antithrombotic activity and method for detecting glycokallidin
US6251393B1 (en) 1998-10-23 2001-06-26 The Brigham And Women's Hospital, Inc. Conformation-specific anti-von Willebrand Factor antibodies
US6605277B2 (en) 1998-10-23 2003-08-12 The Brigham And Women's Hospital, Inc. Conformation-specific anti-von Willebrand factor antibodies
US7112661B1 (en) 1998-10-30 2006-09-26 The Research Foundation Of State University Of New York Variable heavy chain and variable light chain regions of antibodies to human platelet glycoprotein Ib alpha
US6841354B2 (en) * 2000-12-21 2005-01-11 Philadelphia, Health & Education Corporation Screening assay for anti-thrombotic/anti-platelet activity
WO2007077990A1 (fr) 2006-01-06 2007-07-12 Keio University Materiau de distribution de medicament
DE102007031708A1 (de) 2007-07-06 2009-01-08 Dade Behring Marburg Gmbh Bestimmung der von Willebrand Faktor-Aktivität in Abwesenheit von Ristocetin
EP2388596A1 (fr) 2007-07-06 2011-11-23 Siemens Healthcare Diagnostics Products GmbH Procédé pour la détermination de l'activité de clivage du facteur von Willebrand de la protéase ADAMTS-13
US8932820B2 (en) 2007-07-06 2015-01-13 Siemens Healthcare Diagnostics Products Gmbh Methods and kits for determining von Willebrand factor activity in the absence of ristocetin and for determining the activity of ADAMTS-13 protease
US9222942B2 (en) 2007-07-06 2015-12-29 Siemens Healthcare Diagnostics Products Gmbh Methods and kits for determining von willebrand factor activity in the absence of ristocetin
US9869683B2 (en) 2007-07-06 2018-01-16 Siemens Healthcare Diagnostics Products Gmbh Methods and kits for determining von Willebrand factor activity in the absence of ristocetin
US10591490B2 (en) 2007-07-06 2020-03-17 Siemens Healthcare Diagnostics Products Gmbh Methods and kits for determining von willebrand factor activity in the absence of ristocetin

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