[go: up one dir, main page]

WO1992007874A1 - Proteines pharmaceutiquement actives et comprenant une proteine active et une sequence d'affinite a l'integrine - Google Patents

Proteines pharmaceutiquement actives et comprenant une proteine active et une sequence d'affinite a l'integrine Download PDF

Info

Publication number
WO1992007874A1
WO1992007874A1 PCT/GB1991/001860 GB9101860W WO9207874A1 WO 1992007874 A1 WO1992007874 A1 WO 1992007874A1 GB 9101860 W GB9101860 W GB 9101860W WO 9207874 A1 WO9207874 A1 WO 9207874A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
sequence
hirudin
peptide
seq
Prior art date
Application number
PCT/GB1991/001860
Other languages
English (en)
Inventor
Keith Martyn Dawson
Richard Mark Edwards
Anthony Fallon
Original Assignee
British Bio-Technology Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by British Bio-Technology Limited filed Critical British Bio-Technology Limited
Publication of WO1992007874A1 publication Critical patent/WO1992007874A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4732—Casein
    • 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
    • C07K14/75—Fibrinogen
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • compositions comprising an active protein and an integrinaffinity sequence.
  • This invention relates to proteins having a pharmacological activity which may be enhanced by the possession of an integrin affinity site. It also relates to nucleic acids coding for such proteins. In preferred embodiments the invention relates to proteins having antithrombotic activity, such as hirudin, that have an integrin affinity sequence located at the carboxy terminus, and to nucleic acids coding for such proteins.
  • Hirudins are naturally occurring polypeptides of 65 or 66 amino acids in length that are produced by the leech Hirudo medicinalis .
  • Hirudin is an anticoagulating agent which binds to thrombin and prevents blood coagulation by inhibiting thrombin from catalysing the conversion of fibrinogen to fibrin, thus preventing the formation of the protein framework of blood clots.
  • the binding of hirudin also prevents other prothrombic activities of thrombin including activation of factors V, VIII, XIII and platelets.
  • HV-1, HV-2 and HV-3 There are three principal variants of hirudin (named HV-1, HV-2 and HV-3) , whose sequences will be described later.
  • T.J. Rydel et al Science 249: 277-280 (20th July 1990) describes the crystal structure of the complex between recombinant hirudin and thrombin. It describes that the carboxy terminal segment of hirudin makes numerous electrostatic interactions with an anion-binding exosite of thrombin, which is an extension of the active site cleft dominated by positively charged side chains.
  • the carboxy terminal tail of hirudin adopts a long extended confirmation in the complex and is firmly anchored at the end of the thrombin exosite by hydrophobic contacts of the 3 10 helical region (Glu61-Glu65) .
  • hirudin inhibits thrombin by a previously unobserved mechanism.
  • the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. Binding close to the active site involves the three amino-terminal residues of hirudin. Binding to a site distant from the active site (the exosite) involves the carboxy-terminal residues of hirudin.
  • the carboxyl tail of hirudin wraps around thrombin along the putative fibrinogen secondary binding site and makes a number of ionic and hydrophobic interactions with thrombin in this area.
  • integrins as a family of transmembrane glycoproteins which are involved in cell-cell or cell-matrix interactions.
  • Several have been disclosed as binding to extracellular matrix proteins at sites encompassing the Arg Gly Asp (RGD) sequence. These include fibronectin, vitronectin and fibrinogen receptors. Other integrin-ligand binding reactions may not involve RGD sequences.
  • the fibrinogen receptor, ⁇ llb B3, (GpIIb/IIIa) binds fibrinogen, fibronectin, vitronectin and von illebrand factor. When platelets are not activated, the platelet membrane glycoprotein GpIIb/IIIa does not bind fibrinogen. However, upon platelet activation, GpIIb/IIIa becomes competent to bind fibrinogen, a process required for platelet aggregation.
  • integrins as a family of heterodimeric cell surface proteins, each composed of an ⁇ and a ⁇ subunit.
  • the o:IIb/B3 integrin also known as GPIIb/IIIa, binds to fibrinogen, fibronectin, von Willebrand factor and vitronectin.
  • Integrin ⁇ 5B3 binds to the same ligands and also thro bospondin.
  • Integrin ⁇ 2Bl (GPIa/IIa) binds to collagen and laminin.
  • US-A-4703039 describes a method of prolonging the circulatory lifetime of peptides containing a sequence such as RGDS by chemically conjugating them to carrier macromolecules (e.g. albumin) which have the property of an extended lifetime within the circulatory system.
  • carrier macromolecules e.g. albumin
  • EP-A-0333356 (Biogen, Inc.) describes peptides that are from 8 to 26 amino acids in length and homologous to at least a portion of the carboxy terminal 26 amino acids of native hirudin.
  • Such analogues may have an Asp 53 or Asn 53 replaced with an arginine residue so that the peptide contains an Arg 53 -Gly 54 -Asp 55 (RGD) sequence which may bind to a platelet surface glycoprotein (GpIIb/IIIa) and prevent platelet aggregation independently of the thrombin inhibitory action of the peptide.
  • the RGD sequence may therefore target the hirudin peptide to activated platelets.
  • An example of such a peptide that is prepared is N-acetyl-Arg 53 hirudin 53 _ 64 .
  • these short peptides are less active than the full sequence protein (natural hirudin) and have shorter plasma lifetimes.
  • hirudin component was active as the peptide inhibited thrombin and the RGD component was functional as the peptide inhibited integrin-mediated cell adherence.
  • C-terminal peptides of hirudin are less active than the full sequence protein.
  • EP-A-0332533 (Transgene SA) describes hirudin variants that may have the sequence RGDS introduced into a central portion of the molecule, for example hirudin with the variants Arg 33 , Asp 35 and Ser 36 . Also disclosed are hirudin variants that have the C-terminus of the molecule modified by the addition of amino acids, for example proline (such as the introduction of Pro 66 ⁇ ⁇ n order to prevent degradation by carboxypeptidases on expression.
  • proline such as the introduction of Pro 66 ⁇ ⁇ n order to prevent degradation by carboxypeptidases on expression.
  • hirudin is essential for its interaction with thrombin and thus also its antithrombotic activity. It is also known that the presence of an RGD sequence in a protein may or may not confer on that protein the ability to bind to an integrin: it is currently not possible to predict whether an integrin binding sequence, such as RGD, within a protein will be capable of binding to an integrin. It is also known that the pharmacological effectiveness of hirudin, and many other proteins and their variants is dose-dependent and limited by their often short duration of action caused by rapid clearance from the circulation.
  • hirudin which has a short plasma half-life due to rapid renal clearance
  • Kelly et al, Blood 77: 1006-12 (1991) estimated the half-life of hirudin to be 4-5 minutes in baboons and found that intravenous infusion was necessary to inhibit platelet dependent thro botic processes. Consequently, in order to produce prolonged pharmacological activity, e.g. antithrombotic activity as is generally desirable, the protein must be administered by continuous intravenous infusion or repeated subcutaneous injections. Neither of these modes of administration is desirable.
  • a protein comprising a first peptide having pharmacological activity, and a second peptide of no more than fifteen amino acids in length, the second peptide being located at the carboxy terminus of the protein and comprising an integrin affinity sequence, the protein being other than a protein which naturally possesses an integrin affinity sequence at the carboxy terminus.
  • the Applicants have found that placing an integrin affinity sequence at the carboxy terminus of the protein can surprisingly increase the duration of action of the protein, without significantly affecting the protein's biological activity, which may allow fewer and/or lower doses of the protein to be administered.
  • the increased duration of action achieved may also allow administration of the protein by intravenous bolus rather than infusion.
  • the Applicants have also found that the provision of the integrin affinity sequence at the carboxy terminus may also prevent degradation of the protein by carboxypeptidases when the protein is expressed in transformed host cells, thus improving on existing preparative techniques. So, for example, if the protein is synthesised by recombinant DNA technology in transformed yeast cells, there is no need to use mutants deficient in carboxypeptidase activity.
  • the first peptide may be a naturally occurring protein, or a fragment thereof, for example an antithrombotic protein such as tick anticoagulant peptide, antistasin (a leech protein) and in particular hirudin.
  • an antithrombotic protein such as tick anticoagulant peptide, antistasin (a leech protein) and in particular hirudin.
  • Other naturally occurring proteins include a fibrinolytic protein such as tissue plasminogen activator (t-PA) , urokinase or streptokinase; a soluble form of a cell surface receptor used as a receptor by a virus such as soluble CD4; a soluble form of a cell surface receptor such as a cytokine, growth factor, immunoglobulin and/or complement receptor; a growth factor such as epidermal growth factor; a colony stimulating factor (e.g.
  • a cytokine such as interleukin-1; a cytokine inhibitor such as IL-1 inhibitor; a hormone such as growth hormone; an antigen such as an HIV antigen (e.g. HIV p24) ; a soluble form of an adhesin such as ELAM; an enzyme such as asparaginase; an enzyme inhibitor such as alpha-1 antitrypsin; a blood coagulation factor such as factor VII; a neuropeptide such as vasoactive intestinal polypeptide; or a kinin such as bradykinin.
  • a cytokine such as interleukin-1
  • a cytokine inhibitor such as IL-1 inhibitor
  • a hormone such as growth hormone
  • an antigen such as an HIV antigen (e.g. HIV p24)
  • a soluble form of an adhesin such as ELAM
  • an enzyme such as asparaginase
  • an enzyme inhibitor such as alpha-1 antitrypsin
  • a blood coagulation factor such
  • the first peptide may be a variant of a naturally occurring protein, or a fragment thereof, which may differ structurally from, but have similar biological activity to, a naturally occurring protein such as these listed above.
  • the first peptide also encompasses a non-natural protein constructed by protein engineering whose pharmacological activity may or may not be similar to the natural proteins given.
  • fragment when in relation to a fragment of a peptide, it is meant a portion of that peptide that substantially retains at least one (and preferably all) activity of that peptide.
  • variant is meant a polypeptide having one or more amino acids substituted, added or deleted such that the variant substantially retains at least one and preferably all activity of that peptide.
  • amino acid sequence of the first peptide, or a fragment thereof is not altered significantly.
  • pharmacological activity it is meant that the first peptide induces a pharmacological effect on the human or animal to which the protein is administered. Protein sequences which do not induce any biological response or biological or chemical reaction are not included within this definition.
  • Preferred pharmacological activities include antithrombotic and fibrinolytic activities as well as hormonal, growth factor and antigenic activities.
  • the invention has particular application in the preparation of antithrombotic proteins with improved plasma lifetimes, and so the first peptide preferably has antithrombotic activity.
  • the protein of the present invention may thus provide a desired antithrombotic effect and as a result of the presence of the integrin affinity sequence may increase the plasma lifetime of the protein, and so also the length of time over which the protein is active and therefore blood clotting inhibited.
  • the presence of the integrin affinity sequence may provide an additional advantage in that it may serve to target the protein to the site of a blood clot by binding to platelets present at such a clot, for example by binding a receptor such as to the GpIIb/IIIa integrin which is exposed at blood clots on the surface of activated platelets. This binding may thus make such proteins of the present invention more efficient in their antithrombotic activity and may reduce the amount of protein that needs to be administered in order to provide the desired level of inhibition (or even prevention) of blood clotting.
  • the integrin affinity sequence will generally be capable of binding to an integrin.
  • integrin includes receptors (such as cell-surface receptors) and other glycoproteins capable of interacting with ligands. Specifically contemplated are cell surface adhesion receptors that may be capable of mediating a cell-cell and/or cell-extracellular matrix interaction.
  • Suitable receptors include GpIIb/IIIa, vitronectin receptor, fibronectin receptor, fibrinogen receptor, laminin receptor, collagen receptor and von Willebrand receptor.
  • the integrin affinity sequence will generally be capable of binding to an endothelial cell and/or a platelet (for example an activated platelet) .
  • integrin affinity sequences that comprise the sequence RGD (or other sequences, which will be explained later) and/or are specific for integrins that bind ligands possessing the amino acid sequence RGD or other sequences.
  • sequence RGD or other sequences
  • examples of such integrins are the fibronectin, fibrinogen, vitronectin and von Willebrand factor receptors.
  • Other sequences that can be used instead of RGD in the integrin affinity sequence and that are within the scope of the present invention include derivatives of the RGD sequence (such as extended RGD sequences) and sequences where amino acids have been substituted or inverted that are capable of binding integrins. Suitable sequences include:
  • HHLGGAKQAGDV SEQ.ID:7
  • fibrinogen gamma chain platelet integrin affinity sequence 400-411 (Kloczewiak et al, Biochemistry 23:1767-74 (1984));
  • GRGDSP SEQ.ID:8
  • a fibronectin integrin affinity sequence A. Hautenen et al, supra
  • KNQDK (SEQ.ID:9), a kappa casein sequence (EP-A-343085) ;
  • HGDF SEQ.ID:48
  • HQAGDV SEQ. ID: 10
  • DGEA SEQ.ID:20
  • the minimal recognition sequence from collagen required for binding to the collagen receptor ⁇ 2Bl Staatz et al , J. Biol . Chem . 266: 7363-7367 (1991)
  • GREDV SEQ.ID:45
  • REDV SEQ:21
  • CKGDWPC SEQ.ID:23
  • CRGDWPC SEQ.ID:24
  • Cyclic RGD peptides bind to both GPIIb-IIIa and vitronectin receptor while cyclic KGD peptides bind to GPIIb-IIIa but not to vitronectin receptors (WO-A-90/15620) ; or
  • GKDGEA SEQ.ID:46.
  • the integrin affinity sequence may be capable of binding to integrins that are not specific for RGD sequences.
  • An example is the Mac-1 integrin.
  • This integrin is a leukocyte-restricted integrin capable of binding to fibrinogen (in an RGD - independent manner) to a recognition site in fibrinogen not shared with other integrins known to function as fibrinogen receptors (Alteiri D.C. et al , J. Biol . Chem 265: 12119 - 12122 (1990)).
  • the integrin affinity sequence is preferably suitable for binding to an integrin, such as the fibronectin, collagen or fibrinogen receptor. These receptors are glycoproteins that are found on the surface of endothelial cells and platelets.
  • a preferred integrin affinity sequence comprises the sequence -RGDX where X represents nothing or any amino acid. It is preferred that X represent S, F, W or V; S is the amino acid residue of choice. It will thus be seen that the integrin affinity sequence is preferably at least three (such as RGD) , and often four (such as RGDS, RGDF RGDV, RGDW, SGDR, DGEA, REDV and/or KGDW) , amino acids in length.
  • HHLGGAKQAGDV SEQ.ID: 7
  • a five amino acid sequence eg -RGDX- ⁇ X 2 , where X 1 and X 2 independently represent the same as X
  • a six amino acid sequence eg GRGDSP (SEQ.ID:8) , KQAGDV (SEQ.ID:18), CKGDWPC (SEQ.ID:23), GKDGEA (SEQ.ID: 46) and/or CRGDWPC (SEQ.ID:24)
  • Larger integrin affinity sequences such as HHLGGAKQAGDV (SEQ.ID:7), are, however, contemplated.
  • the integrin affinity sequence is preferably from 3 or 4 to 12 or 15 amino acids in length.
  • the second peptide is (i.e. it consists only of) the integrin affinity sequence, although this is not essential.
  • the second peptide is 15 or less, such as 12 or less, and preferably no more than 6, amino acids in length.
  • Proteins which are not encompassed by the present invention are those which, in their natural form, possess an integrin affinity sequence at their carboxy terminus. However, the invention may also exclude any protein which naturally possesses an integrin affinity sequence (anywhere in the protein) , in some embodiments. Such proteins may be (blood) coagulants, or promote blood clot formation. They may also have the integrin affinity sequence HHLGGAKQAGDV (SEQ.ID:7). An example of this is fibrinogen.
  • the protein may have additional peptide sequence(s) placed either at the amino terminus of the first peptide and/or between the first and second peptides.
  • first and second peptides are separate and do not overlap: that is to say one of the peptides does not encompass the other.
  • preferred proteins of the present application may have the following general formula:
  • each of J a and J" individually represent an amino acid (or an amino acid sequence) which may be the same or different;
  • Pep 1 represents the first peptide having pharmacological, such as antithrombotic, activity;
  • Pep 2 represents the second peptide, of up to 15 amino acids in length, comprising an integrin affinity sequence;
  • each of n and m individually represent integers, which may be the same or different, having values of from 0 to 1000.
  • Proteins of the general formula III may be preferred.
  • the peptides (J a ) n arid (J ) m may be any suitable peptide sequences, of any suitable length, from one amino acid upwards provided that they do not substantially interfere with, or inhibit, the integrin affinity of the second peptide (Pep 2 ) . It is also preferred that they do not interfere with, or inhibit, the pharmacological activity of the first peptide (Pep 1 ) .
  • n and/or m preferably has a value of from 0 up to 10, 50, 100 or even 1000.
  • (J a ) n and/or (J b ) m may themselves have antithrombotic activity, and so may be a peptide as defined for the first peptide (Pep 1 ) .
  • (J a ) n and/or (J ) m may comprise a integrin affinity sequence as defined for the second peptide (Pep 2 ) .
  • ( J ⁇ ) ⁇ and/or (J ) m may individually represent a naturally occurring polypeptide, or a variant, or a fragment thereof, having an integrin affinity sequence as discussed before, or may represent fibronectin (or a fragment thereof) .
  • the protein contains one or more integrin affinity sequences in addition to Pep , then one of these sequences is preferably provided at the N-terminus of either (J a ) n or Pep 1 .
  • (J a ) n is provided with an integrin affinity sequence, then this may be provided at the N-terminus. In this situation, it is preferred that the protein does not contain hirudin (or a variant or fragment thereof) .
  • the first peptide (Pep 1 ) can be of any suitable length provided that it possesses pharmacological activity. However, it is preferred that the first peptide is preferably at least 6 (such as at least 8) , amino acids in length.
  • antithrombotic activity in relation to peptides is to be interpreted as that activity possessed by the peptides that are capable of at least partially prohibiting, or reducing, blood clotting; this activity is otherwise known as anticoagulant activity.
  • Preferred peptides have antithrombotic activity. They may therefore be thrombin inhibitors, that is to say they are capable of binding to thrombin so that thrombin does not catalyse the conversion of fibrinogen to fibrin. Such peptides will also usually inhibit, or prevent, the activation of factors V, VIII, XIII and platelets.
  • the first peptide if it has antithrombotic activity, is preferably a naturally occurring antithrombotic peptide, or a fragment thereof (that retains antithrombotic activity) .
  • Hirudin is the preferred peptide.
  • the fragment can be of any suitable length provided that the fragment has antithrombotic activity. Such a fragment may be, for example, a fragment of from 12 to 26 amino acids in length.
  • the fragment preferably corresponds to, or is substantially homologous with, at least a portion of the carboxy terminal 26 amino acids of a naturally occurring protein such as the 26 carboxy terminal amino acids of hirudin.
  • the fragment may correspond to, or be substantially homologous with, at least a portion of the amino terminus of the naturally occurring protein; that is to say, some amino acids at the N-terminus may be deleted while the fragment retains antithrombotic activity.
  • the native full length sequence of the naturally occurring peptide is preferred. For hirudin, this will be 65 or 66 amino acids in length.
  • HV-1 HV-2 and HV-3.
  • HV-l and HV-2 both contain 65 amino acids, while HV-3 has 66.
  • the invention also contemplates a protein comprising a first peptide having pharmacological activity, and an integrin affinity sequence located at the carboxy terminus of the first peptide, other than a protein which naturally possesses an integrin affinity sequence at the carboxy terminus.
  • Proteins in accordance with the first aspect of invention may be prepared by any convenient proce Recombinant DNA technology provides the processes choice.
  • nucleic acid (which may be RNA o preferably, DNA) coding for a protein of the fir aspect.
  • Such nucleic acid is preferably recombina and/or isolated nucleic acid.
  • the nucleic acid may in the form of a vector, such as a plasmid, cosmid virus, and in some embodiments is in an expressib form.
  • a third aspect of the present inventi relates to a vector comprising nucleic acid of t second aspect.
  • the invention also extends to host cells includi mammalian or other animal cells such as insect cell but preferably unicellular organisms, that contain a preferably express, or are capable of expressing, t nucleic acid of the second aspect.
  • a four aspect of the present invention relates to a ho transformed with a vector of the third aspec
  • Unicellular hosts may be eukaryotic or prokaryotic a are preferably mammalian or yeast cells (such as of t genus Saccharomyces or Pi chia .
  • a particular preferred host is a yeast of the species Saccharomyc cerevisiae and/or Pichia pastoris .
  • the host need not be a mutant cell, e.g mutant yea cell with reduced, or without, carboxypeptida activity.
  • Non-mutant yeasts can be used since t integrin affinity sequence can prevent degradation of the protein by any carboxy peptidases present.
  • the nucleic acid of the second aspect may be prepared by chemical synthesis .
  • a large number e . g . a n even numb er s uch a s 8 - 14
  • o f oligonucleotides may be prepared , preferably by chemical synthesis , and then joined together (suitably by kinasing pairs of oligonucleotides and ligating the pairs) .
  • the nucleic acid may be prepared using site directed utagenesis.
  • a nucleic acid primer containing the mutation may be annealed to template nucleic acid containing nucleic acid encoding the protein of the first aspect.
  • the strand containing the primer can be used to prepare double stranded nucleic acid for insertion into a vector (preferably an expression vector) .
  • the vector is preferably capable of replication in both E. coli and yeast (e.g. Saccharomyces cerevisiae .
  • the vector has a selectable marker for maintenance in the yeast host (e.g. Ieu2 gene) and/or an ampicillin resistance locus.
  • the vector also preferably has a promoter, e.g. GAL-10 and/or PGK promoter, for expression.
  • a suitable vector is pSW6 (Accession No. NCIMB 40326) or pJKl.
  • the preferred host is Saccharomyces cerevisiae .
  • proteins of the invention are useful in medicine.
  • a protein in accordance with the first aspect for use in human or veterinary medicine, for example particularly as an antithrombotic agent.
  • a pharmaceutical composition comprising one or more proteins in accordance with the first aspect of the invention and a pharmaceutically or veterinarily acceptable carrier.
  • a composition may be adapted for intravenous administration and may therefore be sterile.
  • compositions in accordance with the invention include preparations of sterile protein(s) of the first aspect in isotonic physiological saline and/or buffer.
  • the composition may include a local anaesthetic to alleviate the pain of injection.
  • the proteins of the invention may be supplied in unit dosage form, for example as a dry powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of protein in activity units. These units may, for example, be antithrombin units (ATUs) , if the protein has antithrombotic activity.
  • ATUs antithrombin units
  • the protein of the first aspect is to be administered by infusion, it may be dispensed by means of an infusion bottle containing sterile water for injection or saline.
  • it may be dispensed with an ampoule of water for injection or saline.
  • the infusible or injectible composition may be made up by mixing the ingredients prior to administration.
  • the amount of protein to be administered will depend on the effect required, such as the amount of antithrombosis, the required speed of action, and the seriousness of a condition of a patient (for example in terms of the extent of clotting, both in the number and size of clots) .
  • the precise dose to be administered will, because of the very nature of the condition which the proteins of the invention are intended to treat, will be determined by the physician.
  • a patient being treated may generally receive a daily dose of from 500 to 50000 ATUs/kg of body weight, for example about 10000 ATUs/kg, either by injection in for example up to five doses, or by infusion.
  • the invention may be used in a method for the treatment or prophylaxis of a human or animal, such as thrombotic disease or disorder, the method comprising the administration of an effective non-toxic amount of a protein of the first aspect.
  • the proteins of the present invention may find use in the treatment of diseases that are caused by either partial or total occlusion of a blood vessel by a blood clot, for example vascular diseases such a myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion and other blood system thromboses.
  • a protein of the first aspect in the preparation of a medicament such as an antithrombotic agent.
  • a process for the preparation of a protein in accordance with the first aspect comprising coupling successive amino acid residues and/or oligo- and/or polypeptides together. This may achieved by chemical synthesis, but is preferably by translation of a nucleic acid of the second aspect in vivo.
  • a process for the preparation of a nucleic acid in accordance with the second aspect of the invention comprising coupling successive nucleotides and/or ligating oligonucleotides and/or polynucleotides together.
  • the nucleic acid may be synthesised chemically, although it is preferred to use a nucleic acid-directed polymerase, preferably in vivo.
  • the nucleic acid may be suitably modified using site-directed mutagenesis.
  • a tenth aspect of the present invention relates to a process for the preparation of a pharmaceutical composition of the sixth aspect, the process comprising admixing a protein of the first aspect with a pharmaceutically or veterinarily acceptable carrier.
  • An eleventh aspect of the present invention relates to a process for the preparation of a vector of the third aspect, the process comprising coupling successive nucleotides and/or ligating oligonucleotides and/or polynucleotides together.
  • a twelfth aspect of the present invention relates to a process for the preparation of a host of the fourth aspect, the process comprising transforming or transfecting a cell with a vector of the third aspect.
  • Figures 1 and 2 are maps of the vectors pSW6 and pJKl respectively used in the Examples and Comparative Examples described later;
  • Figure 3 is an assembly diagram of pairs of annealed oligomers that can be kinased to prepare a synthetic hirudin gene.
  • a synthetic hirudin HV-1 gene was designed incorporating useful unique restriction sites to facilitate manipulation (see SEQ. ID: 4) .
  • the selected codons are favoured by either S. cerevisiae or E. coli and are thus suitable for expression in either organism.
  • the gene sequence was divided into 12 oligodeoxyribonucleotides (see SEQ. ID: 6) . Each oligonucleotide overlapped its adjacent partner by 7 base pairs, thus providing a cohesive end after annealing of complementary pairs of oligonucleotides.
  • the oligonucleotides were synthesised by automated phosphoramidite chemistry on an Applied Systems 380B DNA Synthesiser, using cyanoethyl phosphoramidites. The methodology is now widely used and has already been described (Beaucage, S.L. and Caruthers, M.H. Tetrahedron Letters 24, 245 (1981)).
  • the oligonucleotides were de-protected and removed from the CPG support by incubation in concentrated NH 3 . Typically, 50 mg of CPG carrying 1 micromole of oligonucleotide was de-protected by incubation for 5 hours at 70°C in 600 ⁇ l of concentrated NH 3 .
  • the supernatant was transferred to a fresh tube and the oligomer precipitated with 3 volumes of ethanol. Following centrifugation the pellet was dried and resuspended in 1 ml of water. The concentration of crude oligomer was then determined by measuring the absorbance at 260 nm.
  • the gel was prepared from a stock of 15% acrylamide, 0.6% bisacrylamide and 7M urea in 1 XTBE (90mM Tris-HCl, pH 8.3, 90mM boric acid, 2.5mM EDTA) and was polymerised with 0.1% ammonium persulphate and 0.025% TEMED. The gel was pre-run for 1 hr. The samples were run at 1500 V for 4 to 5 hours. The bands were visualised by UV shadowing and those corresponding to the full length product cut out and transferred to micro-test tubes. The oligomers were eluted from the gel slice by soaking in AGEB (0.5 M ammonium acetate, 0.01 M magnesium acetate and 0.1% SDS) overnight.
  • AGEB 0.5 M ammonium acetate, 0.01 M magnesium acetate and 0.1% SDS
  • the AGEB buffer was then transferred to fresh tubes and the oligomer precipitated with three volumes of ethanol at -70°C for 15 minutes.
  • the precipitate was collected by centrifugion in an Eppendorf microfuge for 10 minutes, the pellet washed in 80% ethanol, the purified oligomer dried, redissolved in 1 ml of water and finally filtered through a 0.45 micron micro-filter. The concentration of purified product was measured by determining its absorbance at 260 nm. d.
  • the oligonucleotides were kinased to provide them with a 5' phosphate thus enabling subsequent ligation. (see Figure 3) .
  • oligomer 100 pmole of oligomer was dried down and resuspended in 20 ⁇ l kinase buffer (70 mM Tris, pH 7.6, 10 mM MgCl 2 , 1 mM ATP, 0.2mM spermidine, 0.5 mM dithiothreitol) .
  • 10 ⁇ l of T4 polynucleotide kinase was added and the mixture was incubated at 37°C for 30 minutes. The kinase was then inactivated by heating at 70°C for 10 minutes.
  • the ligation products were separated on a 2% low gelling temperature agarose gel and the band corresponding to the hirudin HV-l gene was excised and extracted from the gel.
  • the purified fragment was then ligated to Hindlll-EcoRI treated pUC19 plas id DNA.
  • pUC19 code no. ' 27-4951-01, was purchased from Pharmacia Ltd. , Midsummer Boulevard, Central Milton Keynes, Bucks, MK9 3HP, United Kingdom.
  • the transformation of E . coli host strains was accomplished using standard procedures.
  • the strain used as a recipient in the cloning using plasmid vectors was HW87 which has the following genotype:
  • the recombinant pUC19 HV-1 product was transferred into E. coli host strain HW87 and plated onto L-broth ampicillin plates. Twelve colonies were picked and used to prepare plasmid DNA for sequence analysis. Double stranded dideoxy sequence analysis was used to identify a correct clone using a universal sequencing primer BB22 ( CAGGGTTTTCCCAGTCACG) , ( SEQ . ID : 11 ) complementary to the universal primer region of pUC19. The pUC19 recombinant was used to construct the expression vector.
  • An expression vector was designed to enable the secretion of hirudin to the extracellular medium after expression in S. cerevisiae.
  • Secretion of hirudin is desirable to facilitate production of protein with an authentic N-terminus, to ease purification, to limit intracellular proteolysis, to reduce potential toxic effects on the yeast host and to allow optimal protein folding via formation of native disulphide bonds.
  • Secretion of hirudin through the yeast membrane was directed by fusion of hirudin to the yeast mating type alpha-factor pre-pro-peptide (a naturally secreted yeast peptide) .
  • the yeast expression vector pSW6 ( Figure 1) is based on the 2 micron circle from S . cerevisiae . (pSW6 was deposited in _7. cerevisiae strain BJ2168 at The National Collections of Industrial and Marine Bacteria Limited, 23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland, United Kingdom on 23rd October 1990 under Accession No. NCIMB 40326.) pSW6 is a shuttle vector capable of replication in both E . coli and S . cerevisiae and contains an origin of replication for both organisms, the leu2 gene (a selectable marker for maintenance in the yeast host) and the ampicillin resistant locus for selection of plasmid maintenance in E. coli .
  • E. coli contains an alpha- factor pre-pro peptide fused in-frame to epidermal growth factor (EGF) .
  • EGF epidermal growth factor
  • the expression of this fusion is under the control of an efficient galactose regulated promoter which contains hybrid DNA sequences from the S . cerevisiae GAL 1-10 promoter and the S . cerevisiae phosphoglycerate kinase (PGK) promoter.
  • EGF gene in pSW6 can be removed by digestion with Hindlll and BamHI. This removes DNA encoding both EGF and 5 amino acids from the C-terminus of the alpha-factor pro-peptide.
  • Genes to be inserted into the pSW6 expression vector must therefore have the general composition: Hindlll site - alpha-factor adapter - gene - Ba HI site.
  • an oligonucleotide adapter (5' AGCTTGGATAAAAGA 3' (top strand, SEQ.ID:12), 5'TCTTTTATCCA 3' (bottom strand, SEQ.ID:13)) containing part of a Hindlll site and codons encoding the Ser, Leu, Asp, Lys and Arg from the C-terminal end of the alpha factor pro peptide was constructed.
  • the alpha-factor adaptor was ligated to the synthetic HV-l gene such that the recombinant gene encoded an in frame alpha-factor propeptide fusion to hirudin.
  • the pUC19 HV-l plasmid was first cleaved with Bspml. the overhanging ends were next filled with DNA polymerase I to create a blunt ended linear DNA fragment. This fragment was separated from uncut plasmid on a 1% low gelling temperature agarose gel, excised and extracted from the agarose gel matrix, then further treated with Hindlll. The fragment was then ligated to the alpha-factor adaptor (synthesised as two complementary oligonucleotides described above) and annealed prior to ligation.
  • the resultant recombinant plasmid was pJC80.
  • the alpha-factor adaptor - hirudin sequence was removed from pJC80 on a Hindlll-BamHI fragment (SEQ.ID:5).
  • the fragment was purified on a 1% low gelling temperature agarose gel and ligated to Hindlll-BamHI treated pSW6 to create pJKl ( Figure 2) .
  • This plasmid is the basic vector used for wild-type hirudin HV-l expression.
  • Plasmid expression vector pJKl was transformed into yeast (S_. cerevisiae) strain BJ2168 (prc-1-407, prbl-1122 pep4-3 leu2 trpl ura3-52 cir+) using the method of Sherman F. et al (Methods in Yeast Genetics , Cold Spring Harbour Laboratory, (1986)). All yeast media was as described by Sherman et al . Using a 500 ml shake flask, a 100 ml culture of yeast containing pJKl was grown in 0.67% synthetic complete medium yeast nitrogen base, with amino acids minus leucine and 1% glucose as a carbon source.
  • the cells were transferred to a 2 litre shake flask containing 1 litre of the same synthetic complete medium except having 1% galactose and 0.2% glucose as the carbon source. This induces expression from the hybrid PGK promoter. Cells were grown in the induction medium for 3 days. After this period the supernatant was harvested and assayed.
  • Hirudin was purified from yeast culture broth. Cells were first removed by centrifugation. The supernatant was then assayed for biological activity (see Pharmacology Examples) . Production levels from shake flask cultures were routinely between 10-15mg/litre of culture. The hirudin was purified by preparative HPLC (DYNAMAX (Trade Mark) C18, 300 Angstroms pore size, 12 ⁇ m particle size, 21.4 mm internal diameter, 25cm long) . The column was first equilibrated in 15% acetonitrile, 0.1% trifluoro acetic acid. Then up to 21 of supernatant was loaded onto the column with 15% acetroniltrile at lOml/minute.
  • the protein was eluted using a 15-40% acetonitrile gradient at 10ml/minute.
  • the eluted hirudin peak was diluted with an equal volume of 0.1% TFA before loading onto a 10 mm ID DYNAMAX (Trade mark) C18 column at 3 ml/minute.
  • the protein was eluted with a 23-35% gradient.
  • the purity of the isolated protein was assessed by analytical HPLC (VYDAC (Trade Mark) C18 reverse phase) and N-terminal sequence analysis. Mass spectrometry was used to confirm total sequence.
  • N-terminal sequence analysis was performed by automated Edman degradation using an Applied Biosystems Protein Sequencer, model 471 A (Applied Biosystems, Foster City, California) .
  • Hirudin analogues which are altered to include a C-terminal amino acid extension were constructed in order to increase the duration of hirudin antithrombotic activity.
  • Hirudin-RGDS is a hirudin variant in which the amino acid sequence Arg-Gly-Asp-Ser has been added to the C-terminus of hirudin HV-l.
  • the full sequences of hirudin types HV-l, HV-2 and HV-3 with a carboxy terminal -RGDS are given in SEQ. ID.1-3 respectively. The strategy for modification is described below.
  • RZ1032 is a derivative of E. coli that lacks two enzymes of DNA metabolism: (a) dUTPase (dut) which results in a high concentration of intracellular dUTP, and (b) uracil N-glycosylase (ung) which is responsible for removing mis-incorporated uracils from DNA (Kunkel et al, Methods in Enzymol . , 154 , 367-382 ( 1987 ) ) . The principal benef it is that these mutations lead to a higher frequency of mutants in site directed mutagenesis .
  • RZ1032 has the following genotype :
  • JM103 is a standard recipient strain for manipulations involving M13 based vectors.
  • the genotype of JM103 is JM103 delta (lac-pro) , thi r supE.strA. endA. sbcB15, hspR4, F' traD36. proAB. lacl ⁇ . ZdeltaM15.
  • Kinased mutagenesis primer (2.5pmole) was annealed to the single stranded template DNA, which was prepared using RZ1032 as host, (1 ⁇ g) in a final reaction mix of 10 ⁇ l containing 70 mM Tris, 10 mM MgCl 2 .
  • the annealed mixture was then placed on ice and the following reagents added: 4 ⁇ l of 10 X HM (200 mM HEPES, 100 mM MgCl 2 pH 7.6), 5 ⁇ l of a mixture of all 4 deoxyribonucleotide triphosphates each at 5mM, 5 ⁇ l of ATP (lOmM) , 5 ⁇ l DTT (lOOmM) , 2 ⁇ l of T4 DNA ligase (lOOu) , 1.0 ⁇ l Klenow fragment of DNA polymerase and water to a final volume of 50 ⁇ l.
  • the polymerase reaction mixture was then incubated at 15°C for 4-16 hrs. After the reaction was complete, 150 ⁇ l of TE (10 mM Tris, 1 mM EDTA pH 8.0) was added and the mutagenesis mixture stored at -20°C.
  • the mixture was then transformed into the recipient JM103 as follows.
  • a 5 ml overnight culture of JM103 in 2 X YT (1.6% Bactotryptone, 1% Yeast Extract, 1% NaCl) was diluted 1 in a 100 into 50 ml of pre-warmed 2 X YT.
  • the culture was grown at 37°C with aeration until the A600 reached 0.4.
  • the cells were pelleted and resuspended in 0.5 vol of 50 mM CaCl 2 and kept on ice for 15 minutes.
  • the cells were then re-pelleted at 4°C and resuspended in 2.5 ml cold 50 mM CaCl 2 .
  • Single stranded DNA was then prepared from isolated clones as follows: Single plaques were picked into 4 ml of 2 X YT that had been seeded with 10 ⁇ l of a fresh overnight culture of JM103 in 2 X YT. The culture was shaken vigorously for 6 hrs. 0.5ml of the culture was then removed and added to 0.5 ml of 50% glycerol to give a reference stock that was stored at -20°C. The remaining culture was centrifuged to remove the cells and 1 ml of supernatant carrying the phage particles was transferred to a fresh Eppendorf tube. 250 ⁇ l of 20% PEG6000, 250mM NaCl was then added, mixed and the tubes incubated on ice for 15 minutes.
  • the phage were then pelleted at 10,000 rpm for 10 minutes, the supernatant discarded and the tubes re-centrifuged to collect the final traces of PEG solution which could then be removed and discarded.
  • the phage pellet was thoroughly resuspended in 200 mcl of TEN (10 mM Tris, 1 mM EDTA, 0.3 M NaOAc) .
  • the DNA was isolated by extraction with an equal volume of Tris saturated phenol. The phases were separated by a brief centrifugation and the aqueous phase transferred to a clean tube. The DNA was re-extracted with a mixture of 100 ⁇ l of phenol, 100 ⁇ l chloroform and the phases again separated by centrifugation.
  • a gene encoding hirudin RGDS was constructed by oligonucleotide directed mutagenesis.
  • the hirudin gene of Comparative Example 1 was first transferred into M13 mpl9 on a Hindlll-BamHI DNA fragment.
  • the 42bp base pair oligonucleotide CGGGGATCCCTATTAGCTGTCACCGCGCTGCAGATATTCTTC 3 ' (SEQ.ID:17) was used to direct the mutagenesis.
  • Clones carrying the desired mutation were identified by DNA sequence analysis. The entire clone was sequenced to ensure that no other mutation had inadvertently been introduced. After confirmation of the correct DNA sequence on single stranded templates replicative form DNA of one mutant was prepared.
  • pJK009 was transformed first into E. coli host HW87 and characterised by restriction digestion. A 50ml plasmid preparation was prepared and used to transform yeast strain BJ2168.
  • Hirudin-RGDF is a hirudin derivative in which the integrin binding amino acid sequence Arg-Gly-Asp-Phe has been attached to the C-terminus of hirudin HV-l.
  • the procedure of Example 5 was used except that the primer used for mutagenesis was the 42bp primer 5' CGGGGATCCCTATTAGAAGTCACCGCGCTGCAGATATTCTTC 3' (SEQ.ID:15) .
  • EXAMPLE 7 Construction and expression of Hirudin RGDV
  • Hirudin-RGDV is a hirudin derivative in which the integrin binding amino acid sequence Arg-Gly-Asp- Val has been attached to the C-terminus of native hirudin.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 42bp 5' CGGGGATCCCTATTAAACGTCACCGCGCTGCAGATATTCTTC 3' (SEQ.ID:16).
  • Hirudin-RGDW is a hirudin derivative in which the amino acid integrin binding sequence Arg-Gly-Asp-Trp has been attached to the C-terminus of native hirudin.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 42bp 5'CGGGGATCCCTATTACCAGTCACCGCGCTGCAGATATTCTTC 3' (SEQ.ID:14).
  • the C-terminus of recombinant hirudin can be susceptible to proteolysis by carboxypeptidases during production from yeast (R. Bischoff et al, J. Chromatography 476: 245-55, 1989) .
  • Hirudin-RGDF was found to have a mass of 7439.3 for the protonated molecular ion compared to the calculated molecular weight of 7439.
  • the centroid of the molecular ion had a mass of 7476.6-7478.5 compared to the calculated value of 7478.
  • the observed and calculated molecular weights of the hirudin variants are essentially the same indicating that for each variant the C-terminus of the molecule is intact. The addition of an RGDX sequence at the C-terminus of hirudin therefore prevents C-terminal proteolysis.
  • Hirudin-RGDX's are hirudin derivatives in which the amino acid integrin binding sequence RGDX has been attached to the C-terminus of recombinant desulphato-hirudin (X can be any amino acid) .
  • Hirudin variants possessing C-terminal extensions of RGDS (Example 5) , RGDF (Example 6) , RGDV (Example 7) and RGDW (Example 8) have already been described (SEQ.ID NOS:25-28) .
  • Hirudin variants possessing the remaining possible C-terminal amino acids were constructed in order to investigate the effect on the duration of action of antithrombotic activity. Table 1 gives the relationship between the Example number and the integrin affinity sequences (including RGDX sequences) employed.
  • the hirudin-RGDX peptides were constructed and cloned into expression vectors, according to the procedure of Example 5 except that the mutagenesis strategy was complicated by the number of clones sought.
  • the bacteriophage M13 clone containing the hirudin-RGDW DNA sequence of Example 8 was used for the mutagenesis template.
  • RGDR amino acid sequences Arg-Gly-Asp-Arg
  • RGDE Arg-Gly-Asp-Glu
  • RGDC Arg-Gly-Asp-Cys
  • Two specific mutagenic oligonucleotide primers BB5258 28bp (5' GGATCCCTATTAAGCGTCACCGCGCTGC 3') (SEQ.ID NO:52) and BB5259 28bp (5' GGATCCCTATTAGTGGTC- ACCGCGCTGC 3') (SEQ.ID NO:53) were designed and used to construct hirudin variants in which Arg-Gly-Asp-Ala (RGDA) (SEQ.ID NO: 43) and Arg-Gly-Asp-His (RGDH) (SEQ.ID NO:44) respectively, have been added to the C-terminus of hirudin HV-l.
  • RGDA Arg-Gly-Asp-Ala
  • RGDH Arg-Gly-Asp-His
  • the genes encoding the hirudin RGDX variants described above were transferred into expression vectors according to the procedure of Example 5.
  • Hirudin-GREDV is a hirudin derivative in which the amino acid sequence Gly-Arg-Glu-Asp-Val (SEQ.ID NO:45) has been attached to the C-terminus of native hirudin HV-l.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 42bp BB6282 (5'GGATCCCTATTAAACGTCTTCGCGACCCTGCAGATATTCTTC 3') (SEQ.ID NO:54) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.
  • EXAMPLE 27 Construction and Expression of Hirudin-HHLGGAKQAGDV
  • Hirudin-HHLGGAKQAGDV is a hirudin derivative in which the amino acid sequence His-His-Leu-Gly-Gly-Ala-Lys- Gln-Ala-Gly-Asp-Val (SEQ.ID NO:7) has been attached to the C-terminus of native hirudin HV-l.
  • Example 5 The procedure of Example 5 was used except that the oligonucleotide primer comprised a 63bp BB6283 ( 5 ' GGATCCCTATTAAACATCACCTGCCTGTTTTGCACCACCCAGGTGGTGCTGCA- GATATTCTTC 3') (SEQ.ID NO:55) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.
  • Hirudin-CRGDWPC is a hirudin derivative in which the amino acid sequence Cys-Arg-Gly-Asp-Trp-Pro-Cys (SEQ.ID NO:24) has been attached to the C-terminus of native hirudin HV-l.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 48bp BB6284 (5' GGATCCCTATTAACATGGCCAGTCACCGCGACACTGCA- GATATTCTTC 3') (SEQ.ID NO:56) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.
  • Hirudin-CKGDWPC is a hirudin derivative in which the amino acid sequence Cys-Lys-Gly-Asp-Trp-Pro-Cys (SEQ.ID NO:23) has been attached to the C-terminus of native hirudin HV-l.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 48bp BB6285 (5' GGATCCCTATTAACATGGCCAGTCACCCTTACACTGCA- GATATTCTTC 3') (SEQ.ID NO:57) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.
  • Hirudin-GKDGEA is a hirudin derivative in which the amino acid sequence Gly-Lys-Asp-Gly-Glu-Ala (SEQ.ID NO:46) has been attached to the C-terminus of native hirudin HV-l.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 45bp BB6281 (5' GAATCCCTATTATGCTTCACCGTCTTTACCCTGCA- GATATTCTTC 3') (SEQ. ID NO: 58) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.
  • BB6281 contained an undesirable base substitution which was corrected by a further round of mutagenesis with BB6420 22bp (5' GTACCCGGGGATCCCTATTATG 3 ' ) ( SEQ . ID NO : 62 ) .
  • IEGR-Hirudin-RGDW is a hirudin derivative in which the amino acid sequence Ile-Glu-Gly-Arg has been attached to the N-terminus of hirudin-RGDS of Example 5.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 42bp BB4404 (5' GTCGGTGTAAACAACTCTTCCTTCGATTCTTTTATCCAAGCT 3 ' ) (SEQ. ID NO: 59 ) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template .
  • Hirudin [II, T2]-RGDW is a hirudin derivative in which the N-terminus of hirudin-RGDW of Example 8 (Val-Val) was replaced by the amino acid sequence Ile-Thr.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 34bp BB4401 (5' GTACAGTCGGTGTAGGTGATTCTTTTATCCAAGC 3') (SEQ.ID NO:60) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.
  • Hirudin [K24] -RGDW is a hirudin HV-l derivative in which amino acid Gln24 of hirudin-RGDW of Example 8, has been substituted with Lys.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 29bp BB4402 ( 5 ' GCATTTGTTACCTTTACCACAGACGTTAG 3') (SEQ.ID NO:61) and the bacteriophage M13 clone encoding the hirudin-RGDW sequence of Example 8 was used as template.
  • EXAMPLE 34 Construction and Expression of Hirudin [II, T2, K24]-RGDW
  • Hirudin [II, T2, K24]-RGDW is a hirudin derivative in which the N-terminus of hirudin-RGDW of Example 8, (Val-Val) , was replaced by the amino acid sequence Ile-Thr and Gln24 of hirudin-RGDW of Example 8, has been substituted with Lys.
  • the procedure of Example 5 was used except that the oligonucleotide primer comprised a 29bp BB4402 (SEQ.ID NO:61) as used in Example 33 and the bacteriophage M13 clone encoding the hirudin [II, T2]-RGDW sequence of Example 32 was used as template.
  • hirudin and variants were determined. Hirudin samples (50 ⁇ l) diluted in 0.1M Tris HCl pH8.5, 0.15M NaCl, 0.1% PEG 6000 were mixed with 50 ⁇ l human thrombin (Sigma, 0.8U/ml in the above buffer) and 50 ⁇ l CHROMOZYM TH (2.5mM in water) in 96 well plates. The plates were incubated at room temperature for 30 minutes.
  • the reaction was terminated by adding 50 ⁇ l 0.5M acetic acid and the absorbance read at 405nm using an automatic plate reader. Quantitation was performed by comparison with a standard hirudin preparation (recombinant [Lys-47]-HV-2 purchased from Sigma: Sigma Chemical Co. Ltd, Fancy Road, Poole, Dorset BH11 7TG, United Kingdom) .
  • hirudin and the four hirudin-RGD variants is shown in Table 2.
  • the specific activities of hirudin-RGDW, hirudin-RGDS, hirudin-RGDF and hirudin-RGDV determined in this assay were found to be not significantly different from that of unmodified hirudin indicating that the addition of an integrin binding sequence at the C-terminus of hirudin does not impair its activity as a thrombin inhibitor.
  • the antithrombotic activity of the hirudin-RGDS produced using the procedure of Example 5 was measured by determining the time to clot formation using a rat arterio-venous shunt (as described in Markwardt et al Thromb . Haemostasis 47: 226-229 (1982)).
  • Male Sprague-Dawley rats 250 - 400g were anaesthetised with urethane at a concentration of 1.6 g/kg. Through a mid-line incision in the neck, the trachea was cannulated and the animals breathed spontaneously air enriched with oxygen to maintain arterial blood p0 2 between 100 and 150 mmHg.
  • a carotid artery was cannulated for measurement of blood pressure and heart rate.
  • a jugular vein was cannulated for the infusion of supplementary anaesthetic and the antithrombotic protein to be tested, in this case the hirudin-RGDS protein of Example 5.
  • a shunt consisting of two 12.5 cm nylon cannulae, connected by a 2 cm long glass tube 1 mm in diameter, was connected between a carotid artery and a contralateral jugular vein.
  • the shunt was primed with 0.9% saline.
  • a thermistor bead to measure temperature was placed on the surface of the jugular vein nylon catheter, adjacent to the glass section of the shunt. The output of the thermistor was recorded and an output fall indicated that blood flow had ceased and a clot had formed. The time to clot formation was measured from the time to a fall in temperature had occurred after opening of the shunt by removal of the artery clip.
  • Comparative Pharmacology Example B was carried out except using hirudin variant HV-l produced using the procedure of Comparative Examples 1 to 4. This was administered at a dose of 10,000 ATU/kg which resulted in significant prolongation of time to clot formation at 30 minutes after administration of the hirudin to 10.9+1.4 minutes and at 60 minutes to 9.0+1.7 minutes. No significant prolongation was observed at 120 miuntes to 4.9+1.3 minutes after hirudin administration when compared with the control in Comparative Pharmacology Example E.
  • Example F (Applicants') 10.9+1.4 9.0+1.7 4.9+1.3
  • PROPERTIES antithrombotic
  • PROPERTIES antithrombotic
  • PROPERTIES antithrombotic
  • SEQUENCE TYPE nucleotide SEQUENCE LENGTH: 223 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA SOURCE: synthetic FEATURES: hirudin type HV-l gene with 5 amino acid adaptor (corresponding to C- terminus of factor) at amino terminus; from 1 to 6 bp (AAGCTT) is Hindlll site from 118 to 123 bp (GGATCC) is BamI site.
  • STRANDEDNESS double TOPOLOGY: linear MOLECULE TYPE: synthetic DNA SOURCE: synthetic FEATURES: oligomers designed for construction of synthetic type HV-l gene.
  • SEQUENCE TYPE protein SEQUENCE LENGTH: 6 amino acids
  • PROPERTIES integrin affinity sequence in fibronectin
  • PROPERTIES integrin affinity sequence in kappa chain of casein
  • STRANDEDNESS single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA FEATURES: encodes 5 amino acids from carboxy terminus of alpha factor
  • PROPERTIES mutagenesis primer used for hirudin-RGDW
  • PROPERTIES mutagenesis primer used for hirudin-RGDV
  • PROPERTIES mutagenesis primer used for hirudin-RGDS
  • PROPERTIES integrin affinity sequence in fibronectin (REDV)
  • PROPERTIES integrin affinity sequence in snake venom barbourin (KGDW)
  • PROPERTIES integrin affinity sequence selective for GPIIb-IIIa and vitronectin receptor
  • PROPERTIES integrin affinity sequence selective for GPIIb-IIa .(CRGDWPC)
  • SEQUENCE TYPE protein SEQUENCE LENGTH: 4 amino acids
  • PROPERTIES integrin affinity sequence (RGDS)
  • PROPERTIES integrin affinity sequence (RGDF)
  • PROPERTIES integrin affinity sequence (RGDV)
  • SEQUENCE TYPE protein SEQUENCE LENGTH: 4 amino acids
  • PROPERTIES integrin affinity sequence (RGDW)
  • PROPERTIES integrin affinity sequence (RGDR)
  • PROPERTIES integrin affinity sequence (RGDE)
  • PROPERTIES integrin affinity sequence (RGDC)
  • PROPERTIES integrin affinity sequence (RGDK)
  • PROPERTIES integrin affinity sequence (RGDL)
  • PROPERTIES integrin affinity sequence (RGDG)
  • PROPERTIES integrin affinity sequence (RGDM)
  • PROPERTIES integrin affinity sequence (RGDQ)
  • PROPERTIES integrin affinity sequence (RGDT)
  • PROPERTIES integrin affinity sequence (RGDD)
  • PROPERTIES integrin affinity sequence (RGDN)
  • PROPERTIES integrin affinity sequence (RGDY)
  • SEQUENCE TYPE protein SEQUENCE LENGTH: 4 amino acids
  • PROPERTIES integrin affinity sequence (RGDP)
  • PROPERTIES integrin affinity sequence (RGDI)
  • PROPERTIES integrin affinity sequence (RGDA)
  • PROPERTIES integrin affinity sequence (RGDH)
  • PROPERTIES integrin affinity sequence (GREDV)
  • PROPERTIES integrin affinity sequence (GKDGEA)
  • PROPERTIES integrin affinity sequence (SGDR)
  • PROPERTIES mutagenesis primer used for hirudin-RGDR/E/C
  • N G, T, A or C
  • SEQUENCE TYPE nucleotide SEQUENCE LENGTH: 28 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB5258 PROPERTIES: mutagenesis primer used for hirudin-RGDA GGATCCCTAT TAAGCGTCAC CGCGCTGC 28
  • MOLECULE TYPE synthetic DNA : BB5259
  • PROPERTIES mutagenesis primer used for hirudin-RGDH
  • MOLECULE TYPE synthetic DNA : BB6282
  • PROPERTIES mutagenesis primer used for hirudin-GREDV GGATCCCTAT TAAACGTCTT CGCGACCCTG CAGATATTCT TC 42
  • STRANDEDNESS single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB6283 PROPERTIES: mutagenesis primer used for hirudin-
  • STRANDEDNESS single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB6284 PROPERTIES: mutagenesis primer used for hirudin-
  • PROPERTIES mutagenesis primer used for hirudin-
  • STRANDEDNESS single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB6281 PROPERTIES: mutagenesis primer used for hirudin-
  • PROPERTIES mutagenesis primer used for IEGR hirudin- RGDW
  • STRANDEDNESS single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB4401 PROPERTIES: mutagenesis primer used for hirudin [I1,T1] ⁇
  • MOLECULE TYPE synthetic DNA : BB4402
  • PROPERTIES mutagenesis primer used for hirudin[K24]-
  • SEQUENCE TYPE nucleotide SEQUENCE LENGTH: 22 base pairs STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: synthetic DNA : BB6420 PROPERTIES: mutagenesis primer used for hirudin-

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Protéines telles que des analogues de l'hirudine, possèdant une séquence d'affinité à l'intégrine située dans la terminaison carboxy, pouvant présenter des durées de plasma améliorées, cibler des plaquettes dans un caillot sanguin et ne se dégradant pas à l'expression sous l'effet de carboxypeptidases. Les protéines préférées comportent la séquence -RGDX (par exemple, Z= Ser) à la terminaison carboxy.
PCT/GB1991/001860 1990-10-24 1991-10-23 Proteines pharmaceutiquement actives et comprenant une proteine active et une sequence d'affinite a l'integrine WO1992007874A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB909023149A GB9023149D0 (en) 1990-10-24 1990-10-24 Proteins and nucleic acids
GB9023149.9 1990-10-24

Publications (1)

Publication Number Publication Date
WO1992007874A1 true WO1992007874A1 (fr) 1992-05-14

Family

ID=10684292

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1991/001860 WO1992007874A1 (fr) 1990-10-24 1991-10-23 Proteines pharmaceutiquement actives et comprenant une proteine active et une sequence d'affinite a l'integrine

Country Status (7)

Country Link
AU (1) AU8769791A (fr)
GB (1) GB9023149D0 (fr)
IE (1) IE913675A1 (fr)
NZ (1) NZ240307A (fr)
PT (1) PT99293A (fr)
WO (1) WO1992007874A1 (fr)
ZA (1) ZA918423B (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029349A1 (fr) * 1993-06-11 1994-12-22 Merrell Pharmaceuticals Inc. Peptides trifonctionnels antithrombiniques et antiplaquettaires
WO1996034116A3 (fr) * 1995-04-26 1997-01-16 Univ British Columbia Cibles universelles pour identification d'especes
US6451976B1 (en) 1997-03-20 2002-09-17 Trigen Limited Bi-or multifunctional molecules based on a dendroaspin scaffold
WO2009040089A3 (fr) * 2007-09-11 2009-10-29 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2015103643A3 (fr) * 2014-01-06 2015-10-01 The General Hospital Corporation Antagonistes de l'intégrine
JP2018525442A (ja) * 2015-08-05 2018-09-06 シャンシー・ミコ・テクノロジー・リミテッド・カンパニー 抗凝固・抗血小板活性を有するマルチターゲット化合物及びその製法並びに用途
CN113402583A (zh) * 2021-06-19 2021-09-17 江西农业大学 Qgk三肽及其应用
CN113933377A (zh) * 2021-09-28 2022-01-14 深圳湾实验室 一类化合物及其质谱标准品、校正品

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988003810A1 (fr) * 1986-11-27 1988-06-02 Central Blood Laboratories Authority Conjugues pharmaceutiquement actifs presentant une specificite de liaison aux tissus corporels amelioree
EP0311589A1 (fr) * 1987-10-09 1989-04-12 Monsanto Company Activateur tissulaire du plasminogène modifié
EP0332523A1 (fr) * 1988-03-08 1989-09-13 Transgene S.A. Variants de l'hirudine, leurs utilisations et les procédés pour les obtenir
EP0333356A2 (fr) * 1988-03-04 1989-09-20 Biogen, Inc. Peptides de hirudine
WO1990003436A1 (fr) * 1988-09-17 1990-04-05 Basf Aktiengesellschaft POLYPEPTIDES ANALOGUES AU tPA, LEUR FABRICATION ET LEUR UTILISATION
EP0385659A2 (fr) * 1989-03-03 1990-09-05 Fujita Health University Polypeptides multifonctionnels et leur procédé de préparation
EP0207751B1 (fr) * 1985-06-28 1990-11-14 Delta Biotechnology Limited Fibronectines

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0207751B1 (fr) * 1985-06-28 1990-11-14 Delta Biotechnology Limited Fibronectines
WO1988003810A1 (fr) * 1986-11-27 1988-06-02 Central Blood Laboratories Authority Conjugues pharmaceutiquement actifs presentant une specificite de liaison aux tissus corporels amelioree
EP0311589A1 (fr) * 1987-10-09 1989-04-12 Monsanto Company Activateur tissulaire du plasminogène modifié
EP0333356A2 (fr) * 1988-03-04 1989-09-20 Biogen, Inc. Peptides de hirudine
EP0332523A1 (fr) * 1988-03-08 1989-09-13 Transgene S.A. Variants de l'hirudine, leurs utilisations et les procédés pour les obtenir
WO1990003436A1 (fr) * 1988-09-17 1990-04-05 Basf Aktiengesellschaft POLYPEPTIDES ANALOGUES AU tPA, LEUR FABRICATION ET LEUR UTILISATION
EP0385659A2 (fr) * 1989-03-03 1990-09-05 Fujita Health University Polypeptides multifonctionnels et leur procédé de préparation

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029349A1 (fr) * 1993-06-11 1994-12-22 Merrell Pharmaceuticals Inc. Peptides trifonctionnels antithrombiniques et antiplaquettaires
US5681925A (en) * 1993-06-11 1997-10-28 Merrell Pharmaceuticals Inc. Trifunctional antithrombin and antiplatelet peptides
WO1996034116A3 (fr) * 1995-04-26 1997-01-16 Univ British Columbia Cibles universelles pour identification d'especes
US5708160A (en) * 1995-04-26 1998-01-13 The National Research Council HSP-60 genomic locus and primers for species identification
US5989821A (en) * 1995-04-26 1999-11-23 University Of British Columbia Universal targets for species identification
US6451976B1 (en) 1997-03-20 2002-09-17 Trigen Limited Bi-or multifunctional molecules based on a dendroaspin scaffold
WO2009040089A3 (fr) * 2007-09-11 2009-10-29 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2009039976A3 (fr) * 2007-09-11 2009-11-12 Mondobiotech Laboratories Ag Utilisation d'un peptide en tant qu'agent thérapeutique
WO2015103643A3 (fr) * 2014-01-06 2015-10-01 The General Hospital Corporation Antagonistes de l'intégrine
US10533044B2 (en) 2014-01-06 2020-01-14 The General Hospital Corporation Integrin Antagonists
US11939369B2 (en) 2014-01-06 2024-03-26 The General Hospital Corporation Integrin antagonists
JP2018525442A (ja) * 2015-08-05 2018-09-06 シャンシー・ミコ・テクノロジー・リミテッド・カンパニー 抗凝固・抗血小板活性を有するマルチターゲット化合物及びその製法並びに用途
US11643439B2 (en) 2015-08-05 2023-05-09 Shaanxi Micot Technology Limited Company Multi-target compound with anticoagulation and antiplatelet activity, preparation method therefor, and use thereof
CN113402583A (zh) * 2021-06-19 2021-09-17 江西农业大学 Qgk三肽及其应用
CN113933377A (zh) * 2021-09-28 2022-01-14 深圳湾实验室 一类化合物及其质谱标准品、校正品
CN113933377B (zh) * 2021-09-28 2024-08-13 深圳湾实验室 一类化合物及其质谱标准品、校正品

Also Published As

Publication number Publication date
NZ240307A (en) 1993-03-26
GB9023149D0 (en) 1990-12-05
ZA918423B (en) 1993-04-22
PT99293A (pt) 1992-09-30
IE913675A1 (en) 1992-05-22
AU8769791A (en) 1992-05-26

Similar Documents

Publication Publication Date Title
AU644399B2 (en) Proteins and nucleic acids
AU679436B2 (en) Stem cell inhibiting proteins
Fowlkes et al. Potential basis for regulation of the coordinately expressed fibrinogen genes: homology in the 5'flanking regions.
JP3051995B2 (ja) 酸化抵抗性トロンボモジュリン類縁体
AU5675690A (en) Production in bacteria and yeast of hemoglobin and analogues thereof
WO1995017885A1 (fr) Inhibiteurs de l'urokinase activateur du plasminogene
JPS6216931B2 (fr)
CN113493855B (zh) 一种基于RAA-CRISPR-cas13a检测HBV cccDNA的试剂盒
EP0292009A1 (fr) Protéines fibrinolytiques
WO1992007874A1 (fr) Proteines pharmaceutiquement actives et comprenant une proteine active et une sequence d'affinite a l'integrine
CN113201517B (zh) 一种胞嘧啶单碱基编辑器工具及其应用
FI102617B (fi) Ihmisen plasminogeenin aminohappovarianttia koodittava nukleiinihappos ekvenssi, sitä sisältävä ekspressiovektori ja isäntäsolu sekä menetelm ä plasminogeenin aminohappovariantin valmistamiseksi ja menetelmä kaks oiskompleksin muodostamiseksi fibrinolyytt
JP2000508532A (ja) プロリル―４―ヒドロキシラーゼのα２サブユニット、および該サブユニットをコードするプロリル―４―ヒドロキシラーゼ核酸配列およびその製造方法
WO1998051788A2 (fr) Procede et construction pouvant inhiber une migration cellulaire
EP0437367A2 (fr) Compositions et méthodes d'inhibition de l'adhésion des ostéoclastes à l'os
KR20150096019A (ko) 유전자 조작 효율이 향상된 효모 세포 및 그의 용도
EP0381331A2 (fr) Séquences des gènes qui codifient des résidus modifiés situés dans le domaine protéase du t-PA
CN114959108A (zh) 基于RCA-PCR-CRISPR-cas13a检测HBV cccDNA的试剂盒
HU204087B (en) Process for producing humqn alpha or beta interferon
HK1010111A (en) Chimera proteins with fibrinolytic and coagulation inhibiting characteristics
JPH0249586A (ja) 新規血栓溶解剤とその製造方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI JP KR NO US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA