WO2009046742A1 - Variant de staphylokinase - Google Patents

Variant de staphylokinase Download PDF

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Publication number
WO2009046742A1
WO2009046742A1 PCT/EP2007/008871 EP2007008871W WO2009046742A1 WO 2009046742 A1 WO2009046742 A1 WO 2009046742A1 EP 2007008871 W EP2007008871 W EP 2007008871W WO 2009046742 A1 WO2009046742 A1 WO 2009046742A1
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WO
WIPO (PCT)
Prior art keywords
staphylokinase
staphylokinase variant
variant according
variant
amino acid
Prior art date
Application number
PCT/EP2007/008871
Other languages
English (en)
Inventor
Désiré COLLEN
Original Assignee
Thrombogenics N.V.
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 Thrombogenics N.V. filed Critical Thrombogenics N.V.
Priority to TR2010/01630T priority Critical patent/TR201001630T1/xx
Priority to PCT/EP2007/008871 priority patent/WO2009046742A1/fr
Priority to CN200780100736.4A priority patent/CN101848928B/zh
Publication of WO2009046742A1 publication Critical patent/WO2009046742A1/fr
Priority to IL204171A priority patent/IL204171A/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an optimized thrombolytically or fibrinolytically active staphylokinase variant exhibiting low T-cell immunogenicity and reduced clearance by circulating antibodies that can be expressed at high levels.
  • fibrinolytic agents in the early hours following onset of symptoms suggestive of myocardial ischemia preserves myocardial tissue, prevents secondary complications that result from myocardial infarction (congestive heart failure, arrhythmias, etc.), and most importantly - saves lives.
  • the two most commonly used fibrinolytic agents are streptokinase (SK) and recombinant tissue plasminogen activator (rtPA).
  • SK streptokinase
  • rtPA tissue plasminogen activator
  • SK has the advantage of substantially lower cost, it is less effective than rtPA (or mutants of rt-PA) in terms of arterial patency and death at 30 days (and this difference in mortality persists for at least two years) and is associated with hypotension, allergic reactions and neutralizing antibodies.
  • rtPA is not currently the drug of choice for all patients because of its considerable cost and the perceived "marginal" benefit in mortality (an absolute difference of 1% at 30 days).
  • Sak is an enzyme of bacterial origin
  • much effort has been invested in reducing the antigenicity of Sak by site-directed mutagenesis, without reducing the fibrinolytic potency.
  • a Sak variant combining the properties of reduced (T-cell) immunogenicity and reduced clearance (i.e., reduced antibody-binding) that moreover can be produced at levels sufficient to enable broad and wide use.
  • reduced (T-cell) immunogenicity and reduced clearance i.e., reduced antibody-binding
  • the invention relates to an isolated fibrinolytically or thrombolytically active staphylokinase variant characterized in that the amino acid variations introduced in the variant are relative to a staphylokinase, more particularly relative to a wild-type staphylokinase selected from the group consisting of Sak42D, SakC ⁇ c* and SakSTAR, and wherein said amino acid variations comprise one or more of the list of variations selected from E65D, K74Q, R77E, E80S, D82S, V1 12T, K130Y, and E134R. In a particular embodiment the variations comprise V112T and E134R.
  • the staphylokinase variant of the invention consists of the amino acid sequence of SEQ ID NO:2.
  • the additional amino acid variation S3C can be introduced, e.g., to enable modification of the staphylokinase molecule. Said modification can be, e.g., pegylation.
  • an additional N-terminal Methionine residue can be present in the staphylokinases of the invention introduced for, and/or due to, recombinant expression.
  • a next aspect of the invention relates to fibrinolytically or thrombolytically active proteins comprising a staphylokinase variant according to the invention.
  • compositions comprising a staphylokinase variant according to the invention and/or comprising a fibrinolytically or thrombolytically active protein comprising a staphylokinsase variant according to the invention, and comprising at least one of a solvent, diluents or carrier.
  • any of the staphylokinase variants and/or proteins comprising them according to the invention can be used for the manufacture of a medicament for lysing blood clots in a subject.
  • said blood clot can be associated with any thrombotic condition or with catheter occlusion.
  • the invention additionally covers isolated nucleic acids and recombinant vectors comprising a nucleotide sequence encoding a staphylokinase variant according to the invention, optionally operably linked to regulatory nucleotide sequences required for expression of said staphylokinase variant.
  • the invention relates to methods for recombinant production of a staphylokinase variant according to the invention comprising the steps of:
  • said step of modifying said staphylokinase variant consists of pegylation of the staphylokinase molecule.
  • FIGURE 1 Absorption of staphylokinase variants by human antisera.
  • FIGURE 2 In vivo pulmonary embolism lysis after staphylokinase treatment. Lysis of pulmonary embolism in hamsters was measured after treatment by staphylokinase infusion. The proteins were produced and purified concomitantly and the results were obtained in the same series of experiments.
  • Panel A comparison of staphylokinase variant of the invention (SEQ ID NO:1 ; triangles) and wild-type Sak42D staphylokinase (diamonds).
  • Panel B comparison of staphylokinase variant of the invention with the additional pegylated S3C variation (squares) and wild-type SakSTAR staphylokinase (circles). The results demonstrate that the staphylokinase variants of the invention are as efficient as wild-type staphylokinases in this in vivo model.
  • the work leading to the present invention is based in part on staphylokinase amino acid variations as previously disclosed in WO 01/40281 and as published by Warmerdam et al. (J. Immunol. 168, 155-161 (2002); Thromb. Haemost. 87, 666-673 (2002)). These documents highlight the importance of a number of staphylokinase domains (and of individual amino acids within each of these domains) in the elicitation of a T-cell response. Furthermore a number of amino acid variations and combinations of amino acid variations are suggested in order to eliminate much of staphylokinase' s T-cell reactivity.
  • This staphylokinase variant is given in SEQ ID NO: 1 and has the following amino acid variation relative to the wild-type SakSTAR staphylokinase (SEQ ID NO:2), itself initially disclosed by Collen et al. (Fibrinolysis 6, 203-213 (1992)): E65D, K74Q, R77E, E80S, D82S, Vl 12T, K130Y, and E134R.
  • SEQ ID NO:2 wild-type SakSTAR staphylokinase
  • Other wild-type staphylokinases in which the same amino acid variations can be introduced are Sak42D (see DD245444) and SakC ⁇ c* (see EP0077664).
  • an additional amino acid change can be introduced.
  • Pegylation can occur with any suitable type of polythelene glycol (PEG), e.g., PEG-5000 or PEG-20000, and can be performed as described by, e.g., Collen et al. (Circulation 102, 1766-1772 (2000)).
  • PEG polythelene glycol
  • an additional N-terminal Methionine residue can be present in the staphylokinases of the invention introduced for, and due to, recombinant expression.
  • the reduced binding to (pre-existing) antibodies circulating in a subject is important in preventing rapid clearance and/or inactivation/neutralization of staphylokinase when administered to said subject.
  • this property of the staphylokinase variant subject of this invention can limit the amount of active compound that needs to be administered to a subject in need. Inherently this adds to the tolerability and safety of the product, e.g. by preventing an anaphylactic reaction (White, Br.Med.J. 302, 429-430 (1991)).
  • This aspect of the staphylokinase variant of the invention is illustrated in Figure 1.
  • the amino acid variation E65D cannot be derived from any of WO 01/40281 or Warmerdam et al.
  • said degradation included truncation of the amino-terminal serine (or, optionally, cysteine) at position 3, hence precluding pegylation of the staphylokinase variant of the invention additionally bearing the S3C mutation.
  • This problem was solved by the change of a single amino acid, i.e., the change of the wild-type glutamate at position 65 in an aspartate (E65D).
  • the staphylokinase variants of the invention may be incorporated in larger molecules or can be modified (e.g., pegylated) as long as the original immunoreactivity and fibrinolytic or thrombolytic activity of the incorporated staphylokinase variant are conserved.
  • Such larger molecules include, e.g., tagged versions of the staphylokinase variants.
  • the purpose of any of the above changes can, e.g., be to slow down the turnover of the staphylokinase variant once administered to a subject in need.
  • the staphylokinase variant (or modified version thereof) according to the invention will typically be administered to a subject in need in the form of a composition, e.g., a solution.
  • the drug of the invention can be formulated with at least one of a solvent, diluent or carrier.
  • said solvents, diluents or carriers are pharmaceutically acceptable (as can be derived from, e.g., a pharmacopeia).
  • the solvent, diluent or carrier can include any substance enabling efficient administration of the drug to the subject in need, e.g., salts, buffers, (poly)saccharides, (poly)alcohols, DMSO, etc.
  • the staphylokinase variant (or modified version thereof) according to the invention can further be used in the manufacture of a medicament for lysing blood clots in a subject.
  • a subject in need of thrombolytic/fibrinolytic therapy or treatment can suffer from any thrombotic condition such as (acute) myocardial infarction, coronary thrombosis, occlusive stroke, ischemic stroke, deep venous thrombosis, peripheral arterial disease, hepatic vein thrombosis, marasmic thrombosis, sinus thrombosis, venous thrombosis, arterial thrombosis and occlusion of an arterio-venal shunt.
  • the thrombotic condition is occurring during, e.g., infusion of any substance to a patient during which the catheter used for delivering the therapeutic agent(s) is occluded by a blood clot.
  • the staphylokinase variants of the invention can be administered to a subject in need as single or multiple dose(s) or bolus dose(s), by normal infusion, by accelerated infusion, or can be administered proximal to or directly within a thrombus. Combination of any of these administration routes is also an option.
  • staphylokinase variants of the invention can be administered in amounts of 1 ⁇ g to 1 mg/kg body weight, or an amount of 0,1 to 100 mg. This amount can be administered either in a single dose or divided over multiple, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 subsequent doses each interspersed by a suitable amount of time, e.g., 15, 30, or 45 seconds, or from 1 to 10 minutes.
  • the drug is delivered by intravenous or intra-arterial therapy or infusion, or delivered via a catheter to a vascular or coronary thrombus, the time of delivery being dependent on the concentration of the drug in the fluid to be introduced in the vascular system.
  • Typical duration of infusion with a staphylokinase variant of the invention ranges from 5 to 60 minutes.
  • an initial accelerated infusion of 1 to 10 minutes can be followed by normal infusion up to 60 minutes total infusion time.
  • the total dose administered can be in the range of 1 to 100 mg, of 1 to 90 mg, of 1 to 80 mg, of 1 to 70 mg, of 1 to 60 mg, of 1 to 50 mg, of 1 to 40 mg, of 1 to 30 mg, of 1 to 20 mg or of 1 to 10 mg of a staphylokinase variant of the invention.
  • total doses can amount to 0.1, 0.5, 1, 5, 7.5 , 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5 or 100 mg.
  • the total dose of an administered staphylokinase variant of the invention can amount to 0.1, 0.15, 0.3, 0.45, 0.5, 0.6, 0.75, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 4, 5, 6, 7, 7.5, 8, 9 or 10 mg and this total dose can be administered as a single dose or divided over 2, 3, 4 or 5 doses separated by, e.g., 15, 30, or 45 seconds, or by 1 to 10 minutes, e.g., by 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes.
  • the staphylokinase of the invention is typically administered locally into the occluded catheter.
  • the invention further relates to any nucleic acid or recombinant vector (e.g. expression vector) comprising or consisting of a nucleotide sequence encoding a staphylokinase according to the invention.
  • said nucleotide sequence can be operably linked to regulatory nucleotide sequences, e.g., promoter and/or terminator sequences, enabling expression of the encoded staphylokinase. Any suitable combination of regulatory nucleotide sequence(s) and the nucleotide sequence encoding the staphylokinase of the invention is envisaged.
  • the invention relates to methods for recombinant production of a staphylokinase variant according to the invention comprising the steps of:
  • said step of modifying said staphylokinase variant consists of pegylation of the staphylokinase molecule.
  • Non- limiting exemplary host cells include: Escherichia coli, Saccharomyces species (e.g., cerevisiae), Schizosaccharamyces species (e.g., pombe), Hansenula species (e.g., polymorph ⁇ ), insect cells (e.g. Spodoptera frugiperda) and mammalian cells (e.g., COS cells, CHO cells).
  • Staphylokinase variant production Molecular cloning, expression, (recombinant) production and purification of staphylokinase and its variants was performed essentially as extensively described in any of EP0525252 Al, WO 93/13209, WO 96/21016 and WO 99/40198.
  • In vitro fibrinolytic/thrombolytic activity The specific activity of the staphylokinase of SEQ ID NO: 1 in a chromogenic thrombolytic assay was determined as 55 ⁇ 15 kU/mg, this is significantly lower than the activity of wild- type SakSTAR (153 ⁇ 33 kU/mg).
  • the chromogenic assays was performed essentially as described in Example 2.5 of WO 99/40198 using S2403 as chromogenic substrate.
  • concentration of the staphylokinase of SEQ ID NO:1 required to induce 50% clot lysis in human plasma was similar to the required SakSTAR concentration (360 ⁇ 30 ng/niL for SEQ ID NO:1 versus 343 ⁇ 35 ng/mL for SakSTAR).
  • the assay was performed basically as outlined in Amery et al. (Thromb. Diath. Haemorrh. 9, 175-188
  • Thrombolytic Potency The dosages that induced 50% clot lysis were determined from the dose-response curves obtained with TX 174 and SakSTAR in a hamster pulmonary embolism model as described by Stassen et al. (Fibrinolysis 4 (Suppl. 2), 15-21 (1990); Circulation 83 (suppl.IV), IV65-IV72 (1991)). Briefly, a 50- ⁇ L 1251-fibrin-labeled human plasma clot was produced in vitro and injected into the jugular vein of heparinized hamsters.
  • Thrombolytic agents or saline were infused intravenously over 60 minutes, and 30 minutes after the end of the infusion the extent of clot lysis was determined as the difference between the radioactivity initially incorporated in the clot and the residual radioactivity in the lungs and the heart at the end of the experiment.
  • blood samples were collected as described above.
  • the staphylokinase of SEQ ID NO:1 was as potent as SakSTAR in the in- vivo hamster pulmonary embolism animal model (IC50 of 52 ⁇ g/kg for SEQ ID NO:1 versus 55 ⁇ g/kg for SakSTAR). This is further illustrated in Figure 2 where Sak42D (see patent DD245444) was compared with the staphylokinase of SEQ ID NO: 1.

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

Abstract

La présente invention porte sur un variant actif de staphylokinase optimisé thrombolytiquement ou fibrinolytiquement, présentant une faible immunogénicité vis-à-vis des cellules T et une clairance réduite par circulation d'anticorps qui peuvent être exprimés à des niveaux élevés.
PCT/EP2007/008871 2007-10-09 2007-10-09 Variant de staphylokinase WO2009046742A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
TR2010/01630T TR201001630T1 (tr) 2007-10-09 2007-10-09 Stafilokinaz varyantı.
PCT/EP2007/008871 WO2009046742A1 (fr) 2007-10-09 2007-10-09 Variant de staphylokinase
CN200780100736.4A CN101848928B (zh) 2007-10-09 2007-10-09 葡激酶变异体
IL204171A IL204171A (en) 2007-10-09 2010-02-25 An enzyme strain of the staphylocinase family

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2007/008871 WO2009046742A1 (fr) 2007-10-09 2007-10-09 Variant de staphylokinase

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WO2009046742A1 true WO2009046742A1 (fr) 2009-04-16

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IL (1) IL204171A (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102108351B (zh) * 2009-08-19 2013-04-03 河北师范大学 一种低免疫原性葡激酶突变体及其制备方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040198A2 (fr) * 1998-02-04 1999-08-12 Thromb-X N.V. Identification, production et utilisation de derives de la staphylokinase avec immunogenecite et/ou clearance reduites
WO2001040281A2 (fr) * 1999-12-02 2001-06-07 Thromb-X N.V. Procede de réduction de l'immunogénécité de proteines heterologues par l'elimination d'epitopes de lymphocytes t

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040198A2 (fr) * 1998-02-04 1999-08-12 Thromb-X N.V. Identification, production et utilisation de derives de la staphylokinase avec immunogenecite et/ou clearance reduites
WO2001040281A2 (fr) * 1999-12-02 2001-06-07 Thromb-X N.V. Procede de réduction de l'immunogénécité de proteines heterologues par l'elimination d'epitopes de lymphocytes t

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LAROCHE YVES ET AL: "Recombinant staphylokinase variants with reduced antigenicity due to elimination of B-lymphocyte epitopes", BLOOD, vol. 96, no. 4, 15 August 2000 (2000-08-15), pages 1425 - 1432, XP002468295, ISSN: 0006-4971 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102108351B (zh) * 2009-08-19 2013-04-03 河北师范大学 一种低免疫原性葡激酶突变体及其制备方法和应用

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TR201001630T1 (tr) 2010-05-21
IL204171A (en) 2015-05-31
CN101848928B (zh) 2013-07-17
CN101848928A (zh) 2010-09-29

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