EP0707648A1 - Gene de la glycoproteine v des plaquettes et utilisations de ce gene - Google Patents

Gene de la glycoproteine v des plaquettes et utilisations de ce gene

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Publication number
EP0707648A1
EP0707648A1 EP94923370A EP94923370A EP0707648A1 EP 0707648 A1 EP0707648 A1 EP 0707648A1 EP 94923370 A EP94923370 A EP 94923370A EP 94923370 A EP94923370 A EP 94923370A EP 0707648 A1 EP0707648 A1 EP 0707648A1
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EP
European Patent Office
Prior art keywords
leu
gpv
sequence
seq
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP94923370A
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German (de)
English (en)
Inventor
François LANZA
David R. Phillips
Jean-Pierre Cazenave
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COR Therapeutics Inc
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COR Therapeutics Inc
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Priority claimed from US08/195,006 external-priority patent/US6083688A/en
Application filed by COR Therapeutics Inc filed Critical COR Therapeutics Inc
Publication of EP0707648A1 publication Critical patent/EP0707648A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Platelets arise from the fragmentation of megakaryocytes, which are large polyploid bone marrow cells produced by several cycles of chromosomal duplication without cytoplasmic division (Handin (Wilson et. al., eds) in Harrison ' s Principles of Internal Medicine, 12th edition (1991)). Once free of the marrow space, approximately 2/3 of the platelets circulate freely, while approximately 1/3 are sequestered in the spleen. Circulating platelets last for 7 to 10 days, after which they are removed by phagocytic cells. A decrease in platelet mass stimulates megakaryocytopoiesis, resulting in an increase in the number, size and ploidy of the megakaryocytes.
  • Platelet receptors which mediate platelet adhesion and aggregation are located on the two major platelet surface glycoprotein complexes. These complexes are the glycoprotein Ib-IX complex which facilitates platelet adhesion by binding von Willebrand factor (vWF) , and the glycoprotein Ilb-IIIa complex which links platelets into aggregates by binding to fibrinogen.
  • vWF von Willebrand factor
  • vWF von Willebrand factor
  • IIb-IIIa complex which links platelets into aggregates by binding to fibrinogen.
  • Patients with the Bernard-Soulier syndrome, a congenital bleeding disorder show deficient platelet adhesion due to a deficiency in the glycoprotein Ib-IX complex which binds VWF, mild thrombocytopenia, and large lymphocoid platelets.
  • Glycoprotein v is a major ( «-12,000 molecules/ platelet) , heavily glycosylated platelet membrane protein (Mr 82,000) (Modderman et. al. J. Biol . Chem. 267;364-369) .
  • GPIb Consisting of GPIb ⁇ , a 145 kDa protein, disulfide linked to GPIb / 3, a 24 kDa protein
  • GPIX a 22 kDa protein
  • thrombin Since thrombin is now known to activate platelets by cleaving the thrombin receptor (Vu et. al., Cell 64:1057-1068 (1991)), a G-protein coupled receptor, it is unknown whether thrombin cleaves GPV incidently as a consequence of thrombin binding to GPIb ⁇ ;, or whether this cleavage has a physiological role.
  • GPV is also a member of the LRG family (Shimomura et. al., Blood 75:2349-2356 (1990); Roth et. al. , Biochem. Biophys . Res . Commun . 120:153-161 (1990)).
  • GPV is a very specific marker for the megakaryocytic cell lineage.
  • a monoclonal antibody specific for GPV (SW16) was recently shown to bind exclusively to platelets (Modderman et. al., J. Biol . Chem. 267:364-369 (1992)).
  • SWI6 did not bind to red cells, leukocytes, endothelial cells, or cell lines such as HEL or MEG-01 which are known to express platelet megakaryocyte markers.
  • the invention comprises an isolated DNA construct comprising the polynucleotide sequence of the glycoprotein v gene, including the polynucleotide sequence which has the sequence shown in Figure 5A.
  • the polynucleotide sequence encodes a GPV polypeptide, including the amino acid sequence as shown in Figure 5B.
  • the polynucleotide sequence may lack introns, and may incorporate a heterologous promoter operably linked to the polynucleotide sequence which is capable of directing expression in a prokaryote or in a eukaryote.
  • the invention further comprises a DNA construct wherein the polynucleotide sequence encodes a full length glycoprotein V polypeptide.
  • the present application includes prokaryotic or eukaryotic a cell containing a glycoprotein v DNA construct.
  • the present application further provides for a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a glycoprotein v polypeptide.
  • the polypeptide may have the sequence shown in Figure 5B.
  • Figure 1 Cloning, sequencing strategy, and restriction map of a partial human platelet GPV cDNA (a) , and of the complete human GPV gene (b) .
  • the top line represents the coding region (open bar) and 5*-untranslated sequence (hatched bar) for a platelet GPV cDNA with a partial restriction map.
  • the cloning strategy is indicated below. Overlapping clones (i to vi) covering 1,199 bp of cDNA were obtained after PCR amplification of platelet mRNA. The oligonucleotide primers used for the amplification are indicated and the corresponding sequences are listed in Table I.
  • the GPV 8.1 kb genomic fragment (b) was obtained after screening a human genomic library in the ⁇ Fix vector with a 748 bp 32 P-labelled GPV cDNA probe (indicated in (a) by a broken line) .
  • the top line is a partial restriction map of the gene. Exons are boxed: the open box represents the coding sequence, the hatched box represents the 5'-untranslated sequences, and the shaded box represents the 3 •-untranslated region.
  • the vertical arrow indicates the beginning of the genomic sequence reported in Fig. 5A.
  • the open arrowhead indicates the 5*-end and the closed arrowhead indicates the 3•- end of the partial platelet cDNA obtained by PCR.
  • a sequence with perfect consensus for a TATA box is indicated.
  • the closed circles indicate AATAAA consensus sequences for polyadenylation signals.
  • HEL cells with (HEL+PMA) or without (HEL) stimulation with phorbol ester, HL60, and platelet (PLT) RNA were amplified with a GPV primer pair (nt 3,091-3,589) generating a 498 bp band.
  • Figure 3 Northern blot analysis. Total RNA (10 ⁇ g per lane) from human platelets (lanes a and b) and human monocytes (lane c) was electrophoretically separated on 1% agarose-formaldehyde gel, transferred to Zetaprobe, and probed with a 748 bp random prime 32 P-labelled cDNA probe.
  • Lane d is an ethidium bromide stained gel of leukocyte total RNA showing the position of 28S and 18S ribosomal RNA. The molecular size was calibrated by reference to the migration of ⁇ /Hind III DNA fragments indicated in kilo base pairs.
  • Figure 4 Southern blot analysis. High molecular weight genomic DNA 10 vg) from human leukocytes was cut with an excess of Eco RI, Bam HI, and Bgl II restriction endonucleases, separated on a 0.7% agarose gel, and transferred to Hybond N + nylon membranes. The filters were probed with a 748 bp 32 p- labelled GPV cDNA fragment. The size of the hybridizing bands in kilobase pairs was estimated by comparison with ⁇ /Hind III DNA fragments.
  • Figures 5A (SEQ.ID.NO. 1) and 5B (SEQ.ID.NO. 2): Sequence of the human GPV gene Figure 5A and deduced amino acid sequence Figure 5B of the GPV protein.
  • the GPV genomic sequence (SEQ.ID.NO. 1) Figure 5A is shown in the 5 1 - to 3'- orientation with the single intron sequence of 958 bp shown in lower case letters.
  • the gt/ag donor and acceptor sites are in bold characters.
  • Consensus sequences for putative cis-acting promoter elements are indicated as shaded areas.
  • the closed circle indicates a possible Cap site.
  • the ATG translation start and the in-frame TAA stop codon are boxed.
  • the open arrowhead (nt 1,433) and closed arrowhead (nt 3,589) indicate the 5 1 - and 3 1 - end, respectively, of the partial cDNA sequence obtained by PCR amplification of platelet RNA.
  • Possible polyadenylation signal sequences (nt 5,610, nt 6,966, nt 7,224 and, nt 7,358) are double underlined.
  • the putative transmembrane domain is double underlined. Cysteine residues are circled. Potential N-linked glycosylation sites in the extracellular domain are indicated by a vertical arrowhead. N-glycosylation sites that had been identified by protein sequencing are indicated by a star. Internal peptide sequences that were obtained from purified platelet GPV (20, 21), indicated in italics, are underlined by a broken arrow. Differences between the DNA-derived and internal peptides sequences are indicated in parenthesis as lower case letters, (x) indicate a residue which had not been determined in the original peptide sequence.
  • Figure 6 Alignment of the 15 tandem Leu-rich repeated structures for platelet GPV (SEQ.ID.NOs. 22-36). The alignment spans the sequences between residues 61 and 421 of the protein. Identical residues among the 15 segments are boxed. An overall consensus sequence for the GPV repetitive motifs is presented (SEQ.ID.NO. 37).
  • Figure 7 Comparison of the GPV thrombin cleavage site to other thrombin substrates.
  • the GPV sequence around the RG thrombin cleavage peptide bond (SEQ.ID.NO. 38) was aligned with sequences of human fibrinogen (Fg) A ⁇ (SEQ.ID.NOs. 39 and 40) and B ⁇ (SEQ.ID.NO. 41) chains, to human plasma factor XIII (FXIII) (SEQ.ID.NO. 42), and to human chorionic gonatropin ⁇ - subunit (CG3) (SEQ.ID.NO. 43). Amino acid residues identical to GPV are boxed.
  • FIG. 8 Schematic representation of the GPV protein inserted in the platelet plasma membrane in comparison with the GPIb-IX complex.
  • the proteins depicted as bars, are oriented with their NH2- and COOH-termini oriented toward the outside and inside of the cell, respectively. Numbering of amino acids for the mature proteins is indicated.
  • the transmembrane domains are represented as solid rectangles.
  • the Leu-rich (LR) repetitive domains are represented as hatched rectangles.
  • N-glycosylation sites are indicated as solid triangles.
  • GPIb ⁇ contains a region rich in 0-1inked sugars (0- CHO) and is linked to GPIbS by a disulfide (S-S) bond.
  • S-S disulfide
  • the present invention provides the primary structure of the human GPV gene and the structure of the GPV protein.
  • the single-copy gene for GPV is contained within 6.5 kb of genomic sequence, and has a simple structure with a single intron of 958 bp in the 5'-untranslated sequence; the coding sequence is contained within a single exon.
  • the promoter region contains a canonical TATA box, and putative GATA, Ets-1, and Spl cis-acting elements.
  • RT-PCR analysis on RNAs from cells of different hematopoietic origins revealed that GPV was specifically transcribed from platelets and from calls of the megakaryocytic lineage (megakaryocytes, HEL cells) .
  • a single transcript of 4.5 kb for GPV was detected in human platelets by Northern blot analysis, and the entire amino acid sequence of GPV was deduced from the cDNA and genomic sequences.
  • Mature GPV is composed of 543 amino acids which contain a single transmembrane domain, a short cytoplasmic domain (16 residues) and a large extracellular domain with 8 potential N-qlycosylation sites. Analysis of the extracellular domain revealed the presence of 15 tandem Leu-rich repeats of 24 amino acids with homology to GPIb ⁇ , and identified a cleavage site for thrombin near the C-terminus with homology to the A ⁇ chain of fibrinogen.
  • the predicted amino acid sequence of GPV accounts for the known features of the protein. First, it contains with one exception (peptide M4, Shimamura et. al.. Blood 75:2349-2356 (1990) all of the partial peptide sequences which had been reported for purified platelet GPV ( Figure 5A) . Second, the predicted molecular weight of the polypeptide chain of 59,276 Da agrees with the 60 kDa value determined after SDS-PAGE analysis of the deglycosylated protein. Third, the predicted amino acid composition is very similar to that reported for purified GPV when the data are corrected for the 59,276 molecular mass.
  • the LRG repeats in GPV display significant similarity to those found in the subunits of the GPIb-IX complex, which GPV associates with in platelets.
  • the translated protein contains a thrombin cleavage recognition site at a position which would generate a soluble cleavage fragment of the size of GPVfl, a fragment known to be generated after platelet treatment with thrombin (Phillips and Poh-Agin, Biochem. Biophys . Res . Commun . 25:940-947 (1977); Mosher et. al., Blood 52:437-445 (1979)). Analysis of the deduced primary amino acid sequence revealed several distinctive features for GPV.
  • the protein contains an N-terminal signal peptide with a consensus cleavage site (Von Heijne, J. Mol . Biol . 173:243-251 (1984)) -at a Gin residue.
  • N-terminal glutamines are often cyclized to pyroglutamic acids, explaining the N-terminal blockade consistently observed with purified GPV.
  • a second hydrophobic domain was located at the C-terminus of the protein suggesting that GPV is a transmembrane protein. This agrees with data showing that GPV was found in the hydrophobic phase of a Triton X-114 phase partition (Bienz et. al., Blood 6B.:720-725 (1986)).
  • GPV contains 8 potential N-glycosylation sites, located on the extracellular domain.
  • the presence of O-linked carbohydrates and sialic acid has been suggested based upon a 10 kDa molecular weight reduction following neuraminidase treatment (Zafar and Walz Thromb. Res . 52:31-44 (1989)).
  • One short region in the C-terminal region contains two Ser-rich segments and could contain O-linked sugars, but it is probable that the bulk of the carbohydrates are represented by N-sugars due to the observed 20,000 Da apparent molecular weight drop after treatment of GPV by N-glycanase (Zafar and Walz Thromh. Res . 52:31-44 (1989)).
  • GPV has a very short intracellular domain which contains no potential phosphorylation site as it lacks any Tyr, Ser, or Thr residues.
  • the C-terminal intracellular domain also lacks an unpaired cysteine residue, which is a site for acylation by fatty acids which is found in GPIb3 and GPIX (Lopez et. al., Proc. Natl . Acad. Sci . USA 85_:2135-2139 (1988); Hickey et. al. Proc. Natl . Acad. Sci . USA 86:6773-6777 (1989)).
  • GPIb3 and GPIX Lipez et. al., Proc. Natl . Acad. Sci . USA 85_:2135-2139 (1988); Hickey et. al. Proc. Natl . Acad. Sci . USA 86:6773-6777 (1989)
  • GPV has a high affinity binding site for thrombin.
  • the GPVfl fragment is generated at concentrations of thrombin in the nM range: ⁇ -thrombin cleaves 100% of platelet GPV at concentrations less than 30 nM (Jandrot-Perrus et. al. Thromb. Haemostas . 58.:915-920 (1987)).
  • direct interaction of GPV with thrombin was demonstrated by the selective retention of purified GPV on a thrombin-Sepharose column which could then be eluted with heparin (Bienz et. al., Blood 68:720-725 (1986)).
  • platelet proteins known to interact with thrombin with high affinity are the newly cloned thrombin receptor, and GPIb ⁇ (Vu, et. al., Cell 64.:1057-1068 (1991); Lopez et. al., Proc. Nat . Acad. Sci . USA 24:5614-5619 (1987); De marco et. al., J “ . Biol . Chem. 261:23776-23783 (1991)).
  • a distinctive feature of GPV is that it has the highest leucine content (comprising 20% of the amino acids) of the known platelet proteins.
  • LRG domains mediate protein-protein, cell-cell, or cell- matrix interactions.
  • proteoglycan II Korean et. al., Proc. Natl . Acad. Sci . USA 82:7683-7687 (1986)
  • fibromodulin Hamoto et.
  • GPV mRNA in platelets and megakaryocytes were upregulated after treatment with a phorbol ester which is a known inducer of megakaryocyte differentiation in HEL cells.
  • RT-PCR analysis did not reveal GPV mRNA in non megakaryocytic cells such as leukocytes, endothelial cells, HL60 and U937 cells.
  • Northern analysis revealed a transcript of approximately 4.5 kb in platelets and also revealed a positive band of lower size in lymphocytes.
  • the present invention demonstrates that GPV is the product of a single gene.
  • the GPV gene is interrupted by a single intron within the 5'-untranslated region with consensus GT/AG donor and acceptor sites.
  • the GPV gene contains a perfect consensus sequence for a canonical TATA box which is found in the majority of RNA polymerase II transcribed genes. Similar with the other megakaryocyte specific genes, the GPV gene lacks a CAAT sequence, and contains putative binding sites for GATA- 1, Ets-1, and Spl trans-activating factors. Recent experiments support the association of GATA and Ets-1 cis-acting sequences in megakaryocyte-specific gene expression (Lemarchandel et. al., Mol . Cell . Biol . 16:668-676 (1993)) while Spl sites interact with more ubiquitous transcription factors.
  • the availability of the genomic sequence for GPV is useful in the characterization of patients with Bernard-Soulier syndrome. These patients are characterized by an absence of or defect, in the GPIb-IX glycoprotein complex and the GPV platelet glycoprotein.
  • the availability of the GPV cDNA sequence allows for the assessment of the role of GPV in the correct expression of the four proteins which are deficient in the Bernard-Soulier syndrome.
  • the demonstration of a requirement of GPV for correct and efficient formation of the GPIb-IX complex indicates that a defect in the gene for GPV can cause certain types of Bernard-Soulier syndrome.
  • the availability of the genomic sequence allows for the detection of possible alterations in the GPV gene of such patients.
  • 'GPV or "glycoprotein V” refer to polypeptide sequences at least substantially similar to GPV sequence disclosed here. The terms also specifically refer to fragments such as GPVfl as well as the full-length protein. Typically polypeptides will consist of from about 50 to about 560 residues, preferably between about 75 and 500, more preferably between about 100 and about 480 residues.
  • the GPV sequences of the present invention can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art and described in detail, below. For example, the chains can vary from the naturally- occurring sequence at the primary structure level by amino acid insertions, substitutions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
  • the amino acid sequence variants of GPV can be prepared with various objectives in mind, such as facilitating purification and preparation of the protein.
  • the modified molecules are also useful for modifying plasma half life, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use.
  • the amino acid sequence variants are usually predetermined variants not found in nature.
  • the variants typically exhibit the same biological activity as naturally occurring GPV, such as the ability to form complexes with GPIb-IX.
  • variants and derivatives that are not capable of binding to ligands are useful nonetheless (a) as a reagent in diagnostic assays for GPV or antibodies to GPV, (b) as agents for purifying anti-GPV antibodies from antisera or hybridoma culture supernatants when insolubilized in accord with known methods, and (c) as immunogens for raising antibodies to GPV or as immunoassay kit components so long as at least one GPV epitope remains active.
  • modifications of the gene encoding the GPV may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see, Gillman and Smith, Gene 2:81-97 (1979) and Roberts, S.
  • Insertional variants of the present invention are those in which one or more amino acid residues are introduced into a predetermined site in the protein and which displace the preexisting residues.
  • insertional variants can be fusions of heterologous proteins or polypeptides to the amino or carboxyl terminus of GPV. Such fusion proteins can be used to facilitate purification of the encoded protein.
  • Immunogenic fusions may also be produced by cross- linking in vitro or by recombinant cell culture using DNA encoding an immunogenic polypeptide linked to a nucleotide sequence encoding GPV. These immunogenic fusions are useful, for instance, to raise antibodies useful in diagnostics or in purification of GPV by immunoaffinity techniques well known to the skilled artisan. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Non-natural amino acid (i.e., amino acids not normally found in native proteins) , as well as isosteric analogs (amino acid or otherwise) are also suitable for use in this invention.
  • Substantial changes in function or immunological identity are made by selecting substitute residues that differ in their effect on the structure of the polypeptide backbone (e.g., as a sheet or helical conformation), the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain.
  • the substitutions which in general are expected to produce the greatest changes in function will be those in which (a) a hydrophilic residue, e.g. , serine or threonine, is substituted for (or by) a hydrophobic residue, e.g. leucine, isoleucine, phenylalanine, valine or alanine;
  • a cysteine or proline is substituted for (or by) any other residue;
  • a residue having an electropositive side chain e.g., lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, e.g., glutamine or aspartine; or
  • a residue having a bulky side chain e.g.,, phenylalanine, is substituted for (or by) one not having a side chain, e.g. , glycine.
  • Substitutional variants of the subunits also include variants in which functionally homologous (having at least about 70% similarity) domains of other proteins are substituted by routine methods for one or more of the GPV domains.
  • Deletions are characterized by the removal of one or more amino acid residues from the GPV sequence. Deletions of cysteine or other labile residues also may be desirable, for example in increasing the oxidative stability of the protein. Deletion or substitutions of potential proteolysis sites, e.g., Arg Arg, is accomplished by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • a preferred class of substitutional or deletional variants are those involving the transmembrane region of the protein. Inactivation of the transmembrane domain, typically by deletion or substitution of transmembrane domain hydroxylation residues, will facilitate recovery and formulation by reducing its cellular or membrane lipid affinity and improving its aqueous solubility. Alternatively, the transmembrane and cytoplasmic domains can be deleted to avoid the introduction of potentially immunogenic epitopes.
  • Inactivation of the membrane binding function is accomplished by deletion of sufficient residues (not necessarily all the residues) to produce a substantially hydrophilic hydropathy profile at this site or by substituting with heterologous residues which accomplish the same result.
  • transmembrane inactivated GPV A principal advantage of the transmembrane inactivated GPV is that it may be secreted into the culture medium of recombinant hosts.
  • This variant is soluble in body fluids such as blood and does not have an appreciable affinity for cell membrane lipids, thus considerably simplifying its recovery from recombinant cell culture.
  • Deletional variants typically substantially lack a transmembrane domain and consist essentially of the effective portion of the extracellular domain of GPV.
  • the molecule may comprise sequences from the transmembrane region (up to about 10 amino acids) , so long as solubility is not significantly affected.
  • the transmembrane domain may also be substituted by any amino acid sequence, e.g., a random or predetermined sequence of about 5 to 50 serine, threonine, lysine, arginine, glutamine, aspartic acid and like hydrophilic residues, which altogether exhibit a hydrophilic hydropathy profile.
  • a random or predetermined sequence of about 5 to 50 serine, threonine, lysine, arginine, glutamine, aspartic acid and like hydrophilic residues, which altogether exhibit a hydrophilic hydropathy profile.
  • these variants are secreted into the culture medium of recombinant hosts.
  • Glycosylation variants are included within the scope of this invention. They include variants completely lacking in glycosylation (unglycosylated) and variants having at least one less glycosylated site than the native form (deglycosylated) as well as variants in which the glycosylation has been changed. Included are deglycosylated and unglycosylated amino acid sequence variants, deglycosylated and unglycosylated subunits having the native, unmodified amino acid sequence. For example, substitutional or deletional mutagenesis is employed to eliminate the N- or O-linked glycosylation sites of the subunit, e.g. , the asparagine residue is deleted or substituted for by another basic residue such as lysine or histidine.
  • flanking residues making up the glycosylation site are substituted or deleted, even though the asparagine residues remain unchanged, in order to prevent glycosylation by eliminating the glycosylation recognition site.
  • unglycosylated subunits which have the amino acid sequence of the native subunits are produced in recombinant prokaryotic cell culture because prokaryotes are incapable of introducing glycosylation into polypeptides.
  • Glycosylation variants are conveniently produced by selecting appropriate host calls or by in vi tro methods.
  • Yeast for example, introduce glycosylation which varies significantly from that of mammalian systems.
  • mammalian cells from a different species e.g. , hamster, murine, insect, porcine, bovine or ovine
  • tissue than the GPV source are routinely screened for the ability to introduce variant glycosylation as characterized for example by elevated levels of mannose or variant ratios of mannose, fucose, sialic acid, and other sugars typically found in mammalian glycoproteins.
  • In vitro processing of the subunit typically is accomplished by enzymatic hydrolysis, e.g., neuraminidase digestion.
  • polypeptides of the invention can consist of the full length GPV or a fragment thereof as described above.
  • Particularly preferred polypeptides of the invention are those having a polypeptide sequence substantially identical to the sequence disclosed in Figure 5B.
  • Two polynucleotides or polypeptides are said to be "identical” if the sequence of nucleotides or amino acid residues in the two sequences is the same when aligned for maximum correspondence.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math . 1:482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol . Biol . ⁇ :443 (1970), by the search for similarity method of Pearson and Lipman Proc. Proc. Natl . Acad. Sci .
  • the percentage of sequence identity between two sequences is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions. The percentage is calculated by determining the number of positions at which the identical nucleic acid bass or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical means that a polypeptide comprises a sequence that has at least 80% sequence identity, preferably 90%, more preferably 95% or more, compared to a reference sequence over a comparison window of about 20 residues to about 500 residues - typically about 50 to about 500 residues usually about 250 to 300 residues. The values of percent identity are determined using the programs above.
  • polypeptide sequences are substantially identical is if one protein is immunologically reactive with antibodies raised against the other protein.
  • the polypeptides of the invention include polypeptides immunologically reactive with antibodies raised against GPV.
  • the present invention provides substantially pure preparation of GPV polypeptides, produced either by recombinant or synthetic means, or isolated from natural sources.
  • isolated or biologically pure refer to material which is substantially or essentially free from components which normally accompany it as found in its native state.
  • the binding domain polypeptides of this invention do not contain materials normally associated with their m situ environment, e.g., other proteins from a platelet membrane.
  • Isolated polypeptides of this invention do not contain such endogenous co-purified protein. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution will be needed and HPLC or a similar means for purification utilized.
  • Organisms which are commonly utilized as hosts for the amplification of a vector include Escherichia, Bacillus and Streptomyce ⁇ .
  • the most common bacterial hosts are various commercially available strains of E. coli , due to the ease with which the organism may be cultured and the wealth of information which is available regarding the cell's life-cycle, genetics, viruses and developmental regulation.
  • the vectors most commonly used in E. coli are those derived from the pBR322 plasmid and those derived from ⁇ or M13 phage, although several vectors unrelated to any of these are also common.
  • the Sambrook and Berger manuals contains methodology sufficient to direct persons of skill through most cloning exercises.
  • vectors capable of replication in both prokaryotic and eukaryotic cells are generally termed "shuttle vectors" and must contain at a minimum a eukaryotic and a prokaryotic origin of replication.
  • shuttle vectors are commercially available which contain polycloning sites, selectable markers for both bacterial and eukaryotic cells, promoters for both bacterial and eukaryotic expression of the gene(s) of interest, and integration sequences for insertion of the vector into the eukaryotic genome.
  • vectors which may be amplified in bacteria and used for transformation in eukaryotic cells include the family of P element vectors for Drosophila melanogaster , a number of SV40-derived vectors for the transformation of COS cells, adenovirus-derived vectors for transformation in cells containing the appropriate transcription factor for RNA polymerase III, a variety of BPV- derived vectors and the YIp5-derived vectors of Saccharomyces cerevisiae (see Sambrook chapter 16 and Berger chapter 53 for an overview of different vectors which may be transferred between E. coli and eukaryotes) .
  • General techniques for shuttling DNA between prokaryotes and eukaryotes are also described in Cashion et. al., U.S. patent number 5,017,478 and Kriegler, Gene Transfer and Expression : A Laboratory Manual , W.H. Freeman, N.Y. , (1990) which are incorporated by reference.
  • Recombinant proteins may be expressed in either bacteria such as E. coli or in eukaryotic expression systems. In general, it is often necessary to express membrane proteins in eukaryotic systems to achieve proper post-translational modification of the protein, although it is sometimes possible to engineer the biologically active fragment of a polypeptide into an appropriate bacterial expression system, or to use the bacterial system for generating peptides which may be used for antibody generation. In these prokaryotic hosts, one can make expression vectors which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication) .
  • the host cell e.g., an origin of replication
  • promoters or promoter elements will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda (see Yanofsky, C. , 1984, J. Bacteriol . , 158:1018-1024 and Herskowitz, I. and Hagen, D. , 1980, Ann. Rev. Genet . , 14.:399-445) .
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and similar elements for initiating and completing transcription and translation.
  • Methods for expressing large amounts of a protein in a bacterial cell are often invaluable in determining the protein's function, or in generating simple methods of purifying a protein, such as by raising antibodies to a protein expressed in a bacterial cell for use in an immunopurification technique for isolation of a protein from a eukaryotic cell.
  • the expressed polypeptides may first be denatured and then renatured. This can be accomplished by solubilizing the bacterially produced proteins in a chaotropic agent such as guanidine HC1 and reducing all the cysteine residues with a reducing agent such as beta- mercaptoethanol.
  • polypeptides are then renatured, either by slow dialysis or by gel filtration, U.S. Patent No. 4,511,503.
  • the most common of these techniques is the generation of fusion proteins which express a portion of the protein of interest fused to a known antigen which is not otherwise present in the bacterial cell (e.g., LacZ in E. coli) , but for which antibodies are readily available.
  • a known antigen which is not otherwise present in the bacterial cell (e.g., LacZ in E. coli)
  • the fusion protein is used to raise antibodies via standard techniques.
  • genes in eukaryotic systems may be used for a number of purposes, including the following: to confirm the identity of a cloned gene, to express eukaryotic genes which require post-translational modification, to produce large quantities of proteins which are ordinarily available in small quantities from naturally-occurring biological sources, to study the biosynthetic pathway of the gene product, to clarify ' the relationship between the structure and function of a protein through mutational analysis, to properly express proteins containing introns which prokaryotes cannot process, and to identify the gene's promoter elements.
  • Eukaryotic expression vectors contain both prokaryotic origins of replication (generally derived from pBR322) and eukaryotic transcription units which are transcribed only in eukaryotes.
  • the eukaryotic transcription unit consists of non-coding sequences and sequences coding for selectable markers such as thymidine kinase, aminoglycoside phosphotransferase or dihydrofolate reductase, as well as the portion of the gene of interest necessary for expression.
  • selectable markers such as thymidine kinase, aminoglycoside phosphotransferase or dihydrofolate reductase, as well as the portion of the gene of interest necessary for expression.
  • the transcription unit is assembled from well- characterized viral or eukaryotic genes.
  • telomeres may be used to generate cell lines which contain multiple copies of the gene of interest. Call lines with high levels of expression of the introduced gene may be selected by treating the cells with gradually increasing amounts of the toxin which the selectable marker provides resistance against.
  • the DNA unit which is amplified under selective conditions is variable, but generally includes a substantial amount of flanking DNA, particularly in stably transfected lines in which the vector has integrated into the chromosome.
  • the DNA sequences will be expressed in hosts after the sequences have been operably linked to an expression control sequence.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e . g. , U.S. Patent 4,704,362, which is incorporated herein by reference).
  • the vector e.g., a plasmid, which is used to transform the host cell, preferably contains DNA sequences to initiate transcription and sequences to control the translation of the antigen gene sequence. These sequences are referred to as expression control sequences.
  • expression control sequences When the host cell is of insect or mammalian origin, illustrative expression control sequences are obtained from the SV-40 promoter (Science, 222:524-527. 1983), the CMV I.E. Promoter (Proc. Natl . Acad. Sci . 21:659-663, 1984) or the metallothionein promoter (Nature 296:39-42, 1982).
  • the cloning vector containing the expression control sequences is cleaved using restriction enzymes and adjusted in size as necessary or desirable and ligated with D ⁇ A coding for the GPV polypeptide by means well known in the art.
  • polyadenlyation or transcription terminator sequences from known mammalian genes need to be incorporated into the vector.
  • An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (Sprague, J. et al., 1983, J. Virol . 5:773-781) .
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type-vectors. Saveria-Campo, M. , 1985, "Bovine Papilloma virus D ⁇ A a Eukaryotic Cloning Vector” in D ⁇ A Cloning Vol. II a Practical Approach Ed. D.M. Glover, IRL Press, Arlington, Virginia pp. 213-238.
  • the host cells are competent or rendered competent for transformation by various means.
  • the transformed cells are cultured by means well known in the art. Biochemical Methods in Cell Culture and Virology, Kuchler, R.J. , Dowden, Hutchinson and Ross, Inc., (1977) .
  • the expressed GPV polypeptides are isolated from cells grown as suspensions or as monolayers. The latter are recovered by well known mechanical, chemical or enzymatic means. Production of GPV peptides by protein chemistry techniques
  • polypeptides of the invention can be synthetically prepared in a wide variety of ways. For instance polypeptides of relatively short size can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed. , Pierce Chemical Co. (1984). The peptides may be used to generate antibodies using standard methods, including those methods described in this application.
  • purified and isolated GPV may be treated with proteolytic enzymes in order to produce GPV polypeptides.
  • the GPV protein sequence may be analyzed to select proteolytic enzymes to be used to generate polypeptides containing desired regions of the GPV protein.
  • the desired polypeptides are then purified by using standard techniques for protein and peptide purification.
  • standard techniques see, Afeti-tods in Enzymology, "Guide to Protein Purification", M. Deutscher, ed. Vol. 182 (1990), pages 619- 626, which is incorporated herein by reference.
  • Peptides generated by this strategy may be used to generate antibodies using standard methods, including those described in this application.
  • Immunoglobulins are proteins consisting of one or more polypeptides substantially encoded by immunoglobulm genes.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • immunoglobulin forms see e.g., Fundamental Immunology, 2d Ed. W.E. Paul ed. , Raven Press NY (1989), Huston et al., Proc. Nat . Acad. Sci . USA 25:5879-5883 (1988), Bird et al., Science 242:423-426 (1988), and Hunkapiller and Hood, Nature 323:15-16 (1986).
  • immunoglobulin refers to polyclonal antibodies, monoclonal antibodies, to an entire immunoglobulin or antibody or any functional fragment of an immunoglobulin molecule which binds to the target antigen.
  • peptides include complete antibody molecules, antibody fragments, such as Fab, F(ab') 2 , complementarity determining regions (CDRs) , V L (light chain variable region) , V H (heavy chain variable region) , and any combination of those or any other functional portion of an antibody peptide.
  • An F(ab') 2 fragment lacks the C-terminal portion of the heavy chain constant region, and has a molecular weight of approximately 110 kD. It retains the two antigen binding sites and the interchain disulfide bonds in the hinge region, but it does not have the effector functions of an intact IgG molecule.
  • An F(ab') 2 fragment may be obtained from an IgG molecule by proteolytic digestion with pepsin at pH 3.0-3.5 using standard methods such as those described in Harlow and Lane, A ⁇ tiJodies; A Laboratory Manual , Cold Spring Harbor Pubs., N.Y. (1988).
  • An Fab fragment comprises a light chain and the N- terminus portion of the heavy chain to which it is linked by disulfide bonds. It has a molecular weight of approximately 50 kD and contains a single antigen binding site.
  • Fab fragments may be obtained from F(ab') 2 fragments by limited reduction, or from whole antibody by digestion with papain in the presence of reducing agents. (See, Harlow and Lane, supra . )
  • Antibodies which bind to GPV may be produced by a variety of means.
  • the production of non-human monoclonal antibodies, e.g., urine, lagomorpha, equine, etc. is well known and may be accomplished by, for example, immunizing the animal with a preparation containing cells bearing GPV or isolated GPV molecules.
  • Antibody-producing cells obtained from the immunized animals are immortalized and screened, or screened first for the production of antibody which binds to GPV and then immortalized.
  • human monoclonal antibodies to a human antigen is also known in the art. Generation of such human monoclonal antibodies may be difficult with conventional techniques. Thus, it may be desirable to isolate DNA sequences which encode an anti-GPV human monoclonal antibody (or portions thereof) by screening a DNA library from human B cells ⁇ according to the general protocol outlined by Huse et al.. Science 246:1275-1281 (1989). The sequences which encode the antibody (or binding fragment) of the desired specificity are then cloned and amplified.
  • non-human antibodies e.g., the F(ab') 2 or hypervariable regions
  • Fc constant regions
  • the invention also provides synthetic or recombinant immunoglobulins, including chimeric immunoglobulins, humanized antibodies or hybrid antibodies or derivatives of any of those. Chimeric immunoglobulins are typically the product of chimeric DNA, which is recombinant DNA containing genetic material from more than one eukaryotic species.
  • Chimeric immunoglobulins or “chimeric antibodies refer to those antibodies or antibody peptides wherein one portion of the peptide has an amino acid sequence that is derived from, or is homologous to, a corresponding sequence in an antibody or peptide derived from a first gene source, while the remaining segment of the chain(s) is homologous to corresponding sequences of another gene source.
  • a chimeric antibody peptide may comprise an antibody heavy chain with a murine variable region and a human constant region.
  • the two gene sources will typically involve two species, but will occasionally involve different sources from one species.
  • Chimeric antibodies or peptides are typically produced using recombinant molecular and/or cellular techniques.
  • chimeric antibodies typically have variable regions of both light and heavy chains that mimic the variable regions of antibodies derived from one mammalian species, while the constant portions are homologous to the sequences in antibodies derived from a second, different mammalian species.
  • Methods for production of such antibodies are well known and are described in, for example, U.S. Patent No. 4,816,397, and EP publications 173,494 and 239,400, which are incorporated herein by reference.
  • the definition of a chimeric immunoglobulin is not limited to this example.
  • a chimeric antibody is any antibody in which either or both of the heavy or light chains are composed of combinations of sequences mimicking the sequences in antibodies of different sources, whether these sources are differing classes, differing antigen responses, or differing species of origin, and whether or not the fusion point is at the variable/constant boundary.
  • humanized or “human-like immunoglobulin” refers to an immunoglobulin comprising a human-like framework region and a constant region that is substantially homologous to a human immunoglobulin constant region. Hence, most parts of a human-like immunoglobulin, except possibly the CDRs are substantially homologous to corresponding parts of one or more native human immunoglobulin sequences.
  • “Hybrid antibody” refers to an antibody wherein each chain is separately homologous with reference to a mammalian antibody chain, but the combination represents a novel assembly so that two different antigens are recognized by the antibody. In hybrid antibodies, one heavy and light chain pair is homologous to a pair found in an antibody raised against another epitope.
  • Immunoglobulins may be fused to functional regions from other genes (e.g. , those encoding enzymes) to produce fusion proteins (e.g. , immunotoxins) having novel properties.
  • modifications of the genes may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see, Gillman and Smith, Gene, 2:8197 (1979) and Roberts, S. et al, Nature, 222:731-734 (1987)).
  • an immunoglobulin is specific for, or reactive with, a GPV molecule if the immunoglobulin binds GPV as measured or determined by standard antibody- antigen assays, for example, competitive binding assays, saturation assays, or standard immunoassays such as__ELISA or RIA.
  • This definition of specificity applies to single heavy and/or light chains, CDRs, fusion proteins or fragments of heavy and/or light chains, that are also specific for GPV if they bind GPV alone or if, when properly incorporated into immunoglobulin conformation with complementary variable regions and constant regions as appropriate, are then capable of binding GPV.
  • the affinity constant of a specific immunoglobulin of the present invention is typically about 10 "3 to about 10 "12 liters/mole, and preferably about 10 "10 liters/mole or more.
  • binding affinity between two molecules will be influenced by a number of factors such as temperature, pH, ionic strength, and the like.
  • compositions of the present invention comprise immunoglobulins which selectively bind GPV molecules on platelet cells.
  • the immunoglobulins and pharmaceutical compositions of this invention are particularly useful for parenteral administration, i.e., subcutaneous, intramuscular, or intravenous administration.
  • parenteral administration i.e., subcutaneous, intramuscular, or intravenous administration.
  • a number of new drug delivery approaches are being developed, and the pharmaceutical compositions of the present invention are suitable for administration using these new methods, as well. See, Langer, Science, 149:1527-1533 (1990).
  • the antibodies of the present invention can be used to target conventional drugs or other agents to platelets. By using an antibody to target a drug to cells bearing GPV, such drugs can achieve higher concentrations at sites of platelet aggregation.
  • the immunoglobulins can be directly or indirectly coupled to the chemotherapeutic agent.
  • the antibodies of the present invention may also be used for diagnostic purposes, such as identifying areas of platelet aggregation. For diagnostic purposes, the antibodies may either be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the antibody, such as anti ⁇ bodies specific for a particular immunoglobulin constant region. Alternatively, the antibodies can be directly labeled.
  • labels may be employed, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), etc.
  • fluorescers such as fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), etc.
  • enzymes such as fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), etc.
  • ligands particularly haptens
  • compositions containing the immunoglobulins or a cocktail thereof are administered to a patient suspected of having a defect in platelet function.
  • the efficacy of a particular treatment can be monitored.
  • An amount sufficient to accomplish this is defined to be a "diagnostically effective dose.”
  • Kits can also be supplied for use with the subject antibodies.
  • the subject antibody composition of the present invention may be provided, usually in a lyophilized form in a container, either alone or in conjunction with additional antibodies specific for the desired cell type.
  • the antibodies which may be conjugated to a label or toxin, or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like, and a set of instructions for use.
  • buffers such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like
  • these materials will be present in less than about 5% wt. based on the amount of active antibody, and usually present in total amount of at least about 0.001% wt. based again on the antibody concentration.
  • a second antibody capable of binding to the chimeric antibody is employed in an assay, this will usually be present
  • PCR has been adapted for many diverse purposes including cloning, sequencing, forensics, diagnostics and cladistic analysis.
  • the technique is detailed in several general sources which provide adequate guidance to one of skill to perform the technique, including Sambrook and PCR Protocols : A Guide to Methods and Applications (Innis et. al. eds) Academic Press Inc. San Diego, CA (1990) (hereinafter "Innis”) .
  • a 100 ⁇ l reaction contains the following: 1 to 1 x 10 7 target molecules (generally about 1 x 10 5 to 1 x 10 6 target molecules) ; lpmol-lOOpmol of each primer (generally about 20pmol) , the primer having a T m of from about 30°C to about 70°C (preferably greater than about 50°C) 20mM Tris-Hcl (pH approximately 8.3 at 20°C) ; 0.2mM- 5mM MgCl 2 (generally about 1.5 mM MgCl 2 ; occasionally it may be helpful to substitute some of the MgCl 2 with MnCl 2 ) ; 25mM KC1; 0.05% Tween 20; lOO ⁇ g autoclaved
  • reaction mixture is cycled through 15-65 (usually 20-35) of the following temperature variations (generally using a commercially available thermal cycler, occasionally performed by hand with 3 temperature baths): "denaturation” at 96°C for .25 min, (on the first cycle it is often better to leave the reaction mixture at 96°C for 1-5 minutes), "primer annealing” at a temperature about 5°C to 10°C lower than the calculated T m for 30 seconds, “primer extension” at 72°C for 1-3 minutes depending on the length of the target sequence to be amplified.
  • the GPV gene may be examined in patients in order to diagnose Bernard- Soulier syndrome, or in order to determine any genetic predisposition towards the Bernard-Soulier syndrome in persons which may be at risk for the disease.
  • the ability to detect genetic diseases in utero using PCR amplification of DNA from a developing fetus is also known in the art and it will be possible to detect abnormalities in the GPV gene using standard PCR methodologies.
  • the GPV gene is a specific marker for cells of the megakaryocytic lineage and is generally useful as a morphological marker for tracking platelet development.
  • Southern analysis of genomic DNA and northern analysis of RNA using a cloned probe are basic to the art of molecular biology.
  • Sambrook provides adequate guidance to perform most commonly used Southern and northern techniques including analysis of genomic DNA, mRNA and cDNA.
  • the present invention provides an array of probes that may be constructed from the GPV gene for use in Southern analysis. These include synthetic oligonucleotide probes generated from the sequence of any region of the GPV gene, probes generated from cleavage products of the cloned gene using random-primer or terminal phosphate labeling methods and several other methods known to persons of skill.
  • the probes may be used for a variety of purposes including isolation of homologous genes from other species by screening genomic or expression libraries or performing PCR, the identification of restriction fragment length polymorphism and the identification of tissues which express the GPV gene using in situ or northern analysis.
  • the recombinant proteins of the present invention may be used to boost the level of GPV and its cleavage products in a patient which exhibits a lower than normal level of the GPV or GPVfl, or at a specific site such as a wound in a patient with normal clotting.
  • the pharmaceutical compositions of the GPV gene are intended for parenteral, oral, topical, or local administration.
  • the invention provides compositions for parenteral administration that comprise a solution of the GPV polypeptide isolated from an expression system as described above dissolved or suspended in an acceptable aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered, water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium, saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and
  • the GPV polypeptide(s) are preferably supplied in finely divided form along with a surfactant and propellant.
  • the surfactant must be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • a carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
  • the GPV polypeptides may be administered in an aqueous solution as described above, or may be applied in a salve composed of non- toxic carrying agents such as completely polymerized polyacrylamide, or long-chain esters or partial esters of fatty acids as described above. Additionally, the solution or salve may be applied to bandages in standard wound-dressings.
  • GPV polypeptides are administered to a patient in an amount sufficient to affect platelet aggregation, an amount which constitutes a "therapeutically effective dose.” Amounts effective for this use will depend on several factors including the particular polypeptide, the manner of administration, the weight and general state of health of the patient, the presence of other blood disorders in the patient, the presence or severity of a wound and the judgment of the prescribing physician. This will typically be between about 1 ⁇ g/kg and about 100 mg/kg, preferably about 3mg/kg to about 15 mg/kg.
  • Delivery of the polynucleotide of interest may be accomplished in vivo by administering the therapy vector to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) .
  • the vector may be used to deliver polynucleotides to cells ex vivo such as cells explanted from an individual patient or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the gene for the polynucleotide.
  • the vector may be used for gene therapy to treat congenital genetic diseases, acquired genetic diseases (e.g., cancer) , viral diseases or to modify the genome of selected types of cells of a patient for any therapeutic benefit.
  • a treatable disorder using the GPV gene of the present invention is the Bernard-Soulier syndrome.
  • Polynucleotides which reverse or suppress the neoplastic phenotype e.g. antisense inhibition of defective GPV expression
  • Nelly Kieffer Laboratoire Franco- Luxembourgeois debericht Biomedicale, Luxembourg, was from a patient suffering from megakaryoblastic leukemia.
  • Platelet or megakaryocyte polya + RNA was used to synthesize cDNA with a commercial kit (Boehringer Mannheim) .
  • First strand synthesis was performed by priming with oligo dT or by priming with degenerate or exact primers specific for GPV and extending with 20 units of M-MLV reverse transcriptase (Gibco-BRL, Cergy Pontoise, France) .
  • cDNA Approximately 25 ng of platelet or megakaryocyte cDNA was used in the PCR amplification reaction using a Gene Amp DNA amplification reaction kit (Perkin-Elmer Cetus, St. Quentin, France), a 0.2 ⁇ M concentration of each nucleotide primer, and 1 unit of Taq polymerase.
  • the cDNA was denatured at 94°C for 4 min, and amplification was performed for 30 cycles with extension at 72°C for 2 min, denaturation at 94°C for 1 min, and primer annealing between 45 to 60°C for 1 min depending on the primers used.
  • primers 1 and 4 based on peptide sequence K5/6 and running on opposite strands were used successfully to amplify a 108 bp fragment (fragment i) from oligo dT primed platelet cDNA. Sequence analysis revealed that the cDNA fragment contained within primers 1 and 4 coded for a 20 amino acid peptide corresponding exactly to the published peptide sequence (amino acid residues 13 to 33) . This demonstrated that the amplified fragment corresponded to GPV cDNA. In order to obtain additional cDNA sequence, exact oligonucleotide primers were generated in the (-) strand (primer 3) and in the (+) strand (primer 2) orientation.
  • fragment ii An additional 150 bp cDNA fragment (fragment ii) was obtained using primer 3 and degenerate primer 5 based on the M6 peptide sequence. Following PCR, 10 ⁇ l of the amplification mixture was analyzed on a 1 to 2% agarose gel. Rapid amplification of cDNA ends (RACE) was used to extend the sequence in the 5'- and 3'-direction (Frohman, et. al., Proc Natl . Acad. Sci . USA. 25:8998-9002 (1988)). Using the 3'-RACE procedure two additional overlapping fragments (iii and iv) covering 703bp of cDNA were obtained in the 3'- direction.
  • RACE Rapid amplification of cDNA ends
  • cDNA was prepared from 1 ⁇ g of RNA using a (-) strand specific primer and was dG-tailed by incubation with 5 ⁇ M DGTP and 50 units of terminal transferase (Boehringer Mannheim) at 37°C for 10 min in the buffer supplied by the manufacturer. After phenol-chloroform extraction, the reaction mixture was dialyzed over a Centricon 30 column (Amicon, Beverly, MA) and used in the PCR reaction. A first round of PCR was performed with the Adaptor-dC12 primer and primers 7 or 8 followed by a second round of PCR with the adaptor alone and primers 7 or 8.
  • the positive fragments obtained from regular PCR or from the RACE approach were end-cleaved with restriction enzymes (usually Eco RI and Sal I) , isolated by electrophoresis on Sea Plaque agarose (FMC Bioproducts, Rockland, ME) , purified using the Gene Clean II kit, and subcloned into the M13 or pBluescript vectors.
  • restriction enzymes usually Eco RI and Sal I
  • the inserts were sequenced using the Sequenase kit (United States Biochemical Corp. , Cleveland, OH) with DATP 5' ⁇ -[ 35 S]-thiophosphate. All the fragments obtained by the PCR approach were analyzed by sequencing on both strands and their identity to GPV was assessed by comparison to the published GPV partial sequences.
  • EXAMPLE 2 Southern Analysis n ⁇ wnm n Chromosomal DNA
  • a Southern blot analysis was performed under high stringency on human chromosomal DNA using a 748 bp cDNA probe corresponding to the coding region.
  • High molecular weight human leukocyte DNA was digested to completion with restriction endonucleases and subjected to electrophoresis on 0.7% agarose gels. The fragments were transferred to a Hybond N + nylon membrane and were hybridized to a 748 bp 32 P-labelled GPV cDNA fragment at 45°C overnight.
  • the hybridization buffer was 50% (v/v) formamide, 0.9 M NaCl, 50mM NaH 2 P0 4 , 2 mM EDTA, 1% (w/v) SDS, 5% (w/v) dextran sulfate, 0.02% (w/v) polyvinylpyrrolidone, 0.02% (w/v) Ficoll 400, and 50 ⁇ g/ml salmon sperm DNA.
  • Membranes were washed in 0.5X SSC, 1% (w/v) SDS at 60°C and autoradiographed.
  • genomic clones were isolated from a human fibroblast genomic library in the phage ⁇ Fix vector.
  • Approximately 8 x 10 5 recombinants of a human commercial genomic library in the ⁇ Fix vector (Stratagene) were plated on E. coli LE392, transferred to nitrocellulose membranes, and probed with a 748 bp ⁇ p labelled GPV cDNA fragment.
  • the hybridization conditions were 50% (v/v) formamide, 5X SSC, 0.1% (w/v) SDS, 5X Denhardt's medium, and 0.1 mg/ml salraon sperm DNA at 42°C overnight.
  • the filters were washed in 0.1X SSC, 0.05% (w/v) SDS at 56°C, dried and exposed for autoradiography. Positive clones were subjected to two additional rounds of screening in order to obtain isolated clones.
  • Phage DNA was purified using the liquid lysis procedure. The DNA was digested with EcoRI, separated on a 0.7% agarose gal, transferred to nitrocellulose, and hybridized to the 32 p-labelled GPV cDNA fragment to localize exon containing fragments. The positive fragments were subcloned into the pBluescript vector for further restriction enzyme analysis, and finally subcloned into the M13 sequencing vector. After characterization by restriction endonuclease mapping and Southern blot analysis, clone G5a was chosen for further subcloning, restriction enzyme analysis, and nucleotide sequencing.
  • the sequence immediately adjacent to the 5'-end of the cDNA (exon 1) was examined for the presence of cis- regulatory elements.
  • the analysis revealed the presence of a sequence which matched the consensus sequence for a TATA box (5'-TATATA-3 ') , characteristic of RNA polymerase II transcribed genes, but did not reveal a consensus sequence for a CAAT box.
  • the TATA box was followed 31 bp downstream by a putative Cap site.
  • An additional sequence (TATAT) with similarity to the TATA box consensus was found at position 1,199.
  • a 5*-AAGATA-3' and a 5•-AGATAG-3' sequence with similarity to the consensus 5'-(AT)GATA(AG)-3• motif for a GATA-1 binding site were located at position 1,285 and 1,321 respectively.
  • the GATA motif has been found in the promoters and enhancers of all characterized erythroid and megakaryocyte specific genes.
  • motifs for cis-acting elements include Ets-1 cis-acting sequences at positions 471 (5'-CAGGAAGT-3•) , 493 (5'-GAGGAAGC-3') , 897 (5'- GCATCCTG-3', inverse), 1,178 (5'-ACTTCCC-3 ' , inverse) and, 1,365 (5'-CAGGATGCAA-3*) (SEQ.ID.NO 3) (consensus sequence: 5'-(GC) (AC)GGA(AT)G(TC) ) , and a Spl putative binding site at position 1,142 (5'-GGGGTGTGGC-3*) (SEQ.ID.NO.
  • EXAMPLE 5 Determination of The Primary Amino Acid Structure of GPV
  • the amino acid sequence of GPV as deduced from its cDNA and genomic sequences is shown in Figure 5A (SEQ.ID.NO. 2) .
  • GPV was found to be composed of 560 amino acids, including a putative 16 amino acid signal peptide, and a putative C- terminal 25 amino acid transmembrane domain. Between the signal peptide and the transmembrane domain, is a sequence of 503 amino acids containing eight potential N-glycosylation sites (NXS, NXT) and eight cysteine residues. The putative transmembrane domain is followed by a 16 residue hydrophilic segment.
  • the carboxy region of the transmembrane domain contains basic residues which are typically found on the cytoplasmic side of the integral membrane proteins (Sabatini et. al., J. Cell . Biol . 21:1-22 (1982)). These features suggest that GPV is a type I integral membrane protein with most of its polypeptide chain located outside the call ( Figure 8) . The predicted molecular weight of the GPV polypeptide after removal of the signal peptide is 59,276 Da.
  • Thrombin-induced cleavage of GPV results in the generation of a soluble fragment (GPVfl) of approximately 69 kDa.
  • GPVfl soluble fragment
  • a sequence was found containing an RG dipeptide which represents a potential cleavage site for thrombin (Stubbs and Bode Thromb. Res . 2 :1-58 (1993)). Proteolytic cleavage at this RG site would cause a 67,613 Da loss in the molecular weight of GPV.
  • GPV mRNA The cellular distribution of GPV mRNA was assessed using the sensitive RT-PCR amplification technique using primers from the cDNA sequence. GPV mRNA was detected in platelets, megakaryocytes and HEL cells, and was increased in HEL cells after stimulation with phorbol ester, but was not detected in HL60 cells, K562, U937, or endothelial cells (Figure 2) .
  • MOLECULE TYPE DNA (genomic)
  • CTGCACCCCA GCCTGGCGAC AGAGTCCCCC TCCCCCACCA AAAAAACAAC AAGTGAGCAT 900
  • CTCTGTGGCC CAGGCTGGCG TGCAGTGGGC CGTCTCAGTT CACTGCAGCC TCCGCCCTCC 4731
  • Primer 7 (-) 5' -TAT CAG GTC ACT GAA GGT GCC-3' SEQ.ID.NO.
  • Primer 8 (-) 5' -AAG ACA CAC TTG CAA GCT (SEQ.ID.NO.
  • the K/6 and M6 peptide sequences were taken from ref.X and numbered accordingly.
  • Eco RI and Sal I restriction sites we added at the 5' -end of coding (+) and non coding (-) strand primers respectively to facilitate further subcloning of th PCR products.

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  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
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  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne le gène de la glycoprotéine v, et révèle en particulier la séquence et la structure de ce gène, ainsi que la séquence d'acides aminés du polypeptide de la glycoprotéine v. La relation évolutive du gène de la glycoprotéine v avec d'autres glycoprotéines est également décrite, et plusieurs utilisations de ce gène isolé sont proposées.
EP94923370A 1993-07-09 1994-07-07 Gene de la glycoproteine v des plaquettes et utilisations de ce gene Withdrawn EP0707648A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US8945593A 1993-07-09 1993-07-09
US89455 1993-07-09
US16259993A 1993-12-03 1993-12-03
US162599 1993-12-03
US08/195,006 US6083688A (en) 1993-07-09 1994-02-10 Platelet glycoprotein V gene and uses
PCT/US1994/007644 WO1995002054A2 (fr) 1993-07-09 1994-07-07 Gene de la glycoproteine v des plaquettes et utilisations de ce gene
US195006 2000-04-06

Publications (1)

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EP0707648A1 true EP0707648A1 (fr) 1996-04-24

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EP94923370A Withdrawn EP0707648A1 (fr) 1993-07-09 1994-07-07 Gene de la glycoproteine v des plaquettes et utilisations de ce gene

Country Status (6)

Country Link
EP (1) EP0707648A1 (fr)
JP (1) JPH09500017A (fr)
AU (1) AU7325794A (fr)
CA (1) CA2166872A1 (fr)
SG (1) SG70981A1 (fr)
WO (1) WO1995002054A2 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6225085B1 (en) * 1998-04-21 2001-05-01 Millennium Biotherapeutics, Inc. LRSG protein and nucleic acid molecules and uses therefor
US7033790B2 (en) 2001-04-03 2006-04-25 Curagen Corporation Proteins and nucleic acids encoding same
EP3184149A1 (fr) * 2015-12-23 2017-06-28 Julius-Maximilians-Universität Würzburg Glycoprotéine v soluble pour le traitement de maladies thrombotiques
EP3184544A1 (fr) * 2015-12-23 2017-06-28 Julius-Maximilians-Universität Würzburg Inhibiteurs de la glycoprotéine v pour une utilisation comme coagulants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9502054A2 *

Also Published As

Publication number Publication date
WO1995002054A3 (fr) 1995-06-08
SG70981A1 (en) 2000-03-21
JPH09500017A (ja) 1997-01-07
CA2166872A1 (fr) 1995-01-19
WO1995002054A2 (fr) 1995-01-19
AU7325794A (en) 1995-02-06

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