WO1995018827A1 - Derives du facteur viii - Google Patents

Derives du facteur viii Download PDF

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Publication number
WO1995018827A1
WO1995018827A1 PCT/DK1995/000008 DK9500008W WO9518827A1 WO 1995018827 A1 WO1995018827 A1 WO 1995018827A1 DK 9500008 W DK9500008 W DK 9500008W WO 9518827 A1 WO9518827 A1 WO 9518827A1
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WO
WIPO (PCT)
Prior art keywords
factor viii
ser
leu
val
glu
Prior art date
Application number
PCT/DK1995/000008
Other languages
English (en)
Inventor
Else Marie Nicolaisen
Egon Persson
Mirella Ezban Rasmussen
Original Assignee
Novo Nordisk A/S
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 Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU13825/95A priority Critical patent/AU1382595A/en
Publication of WO1995018827A1 publication Critical patent/WO1995018827A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • 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 Factor VIII derivatives comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues, said Factor VIII derivatives showing a coagulant activity of Factor VIII, a method for the preparation of Factor VIII derivatives comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues.
  • the invention relates to pharmaceutical preparations comprising a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues substituted by a serine residue, and the use of a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues for the preparation of a pharmaceutical preparation for the treatment of diseases caused by an absence or deficiency of the Factor VIII of a subject.
  • Haemophilia A is an X-chromosome-1inked inherited disease which afflicts 1-2 males per 10,000. The disease is caused by an absence or deficiency of Factor VIII.
  • Factor VIII is a large glycoprotein (native M r 330000 - 360000), which is present in plasma at low concentrations (0.1 nM (Rapaport, West.J.Med. (1993) 158.153-161)). It is an essential element in the proteolytic cascade which converts soluble fibrinogen to insoluble fibrin, forming a clot to prevent blood loss from traumatized tissue. In the bloodstream, it is found in noncovalent association with von Willebrand factor (vWF) which acts as a stabilizing carrier protein.
  • vWF von Willebrand factor
  • Factor VIII is susceptible to cleavage by thrombin, activated protein C, plasmin, and other serine proteases. It is generally isolated from plasma or plasma products as a series of related polypeptides ranging from M r 160000-40000 with predominant species of M r 92000 and M r 80000-77000. This complex pattern has made the analysis of the structure of active Factor VIII very difficult.
  • the full-length protein contains three repeats of the A-domain and two repeats of the C-domain together with a heavily glycosylated B-domain, ordered A1-A2-B-A3-C1-C2.
  • the B-domain is not required for the function of Factor VIII (Burke et al. (1986) J. Biol. Chem. 261:12574-12578).
  • the heavy chain is cleaved between the Al and the A2-domains C-terminal of Arg-372 and 41 amino acids are cleaved off from the N-terminus of the light chain C-terminal of Arg 1688.
  • Factor VIII has historically been isolated from blood in a concentrated form for therapeutic treatment of haemophilia. However, Factor VIII is only present in the blood in extremely small amounts and a vast number of donors have to be involved. Moreover, the purification process is laborious and expensive. Concerns regarding transmission of HIV and other blood-borne diseases as well as shortage of supplies have especially stimulated activity to provide alternative supplies of Factor VIII, thus leading to the development of recombinant techniques.
  • Factor VIII in order to ensure that as few units of Factor VIII as possible are lost during activation as well as storing and handling of Factor VIII and pharmaceutical preparations comprising the same.
  • Cys-amino acids may be replaced by another amino acid, preferably serine, when combining an N-terminal fragment of Factor VIII:C with a molecular weight of 92 to 210 kD and a C-terminal fragment of Factor VIII:C with a molecular weight of 80 to 70 kD
  • the present invention relates to a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by one or more other amino acid residues, said Factor VIII derivative showing a coagulant activity of Factor VIII.
  • the invention relates to a method for the preparation of Factor VIII derivatives comprising a
  • the invention relates to pharmaceutical preparations comprising a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues.
  • the invention relates to the use of a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues for the preparation of a pharmaceutical preparation.
  • the present invention relates to a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues, said Factor VIII derivative showing a coagulant activity of Factor VIII.
  • a cysteine residue is substituted by an amino acid residue selected from the group consisting of alanine, threonine, serine, glycine and asparagine, more preferred by a serine residue.
  • the coagulant activity is in the same order of magnitude as the coagulant activity of the corresponding "unsubstituted" Factor VIII and is maintained for an extended period of time as compared with the very rapid decline of the coagulant activity of Factor VIII after activation.
  • the amino acid sequence of the Factor VIII derivatives of the invention may correspond to the amino acid sequence of full length human Factor VIII as described above.
  • the Factor VIII derivative may also have an amino acid sequence corresponding to a shortened form of Factor VIII having one or more deletions in the molecule, subunits of Factor VIII or complexes of subunits of Factor VIII provided that the Factor VIII derivative comprises the parts of the Factor VIII molecule being necessary for imparting the molecule coagulant activity per se or after activation using e.g. thrombin.
  • a complex may be held together by a ionic bridge or any other chemical binding imparting the complex coagulant activity.
  • a ionic bridge may be a divalent metal bridge comprising e.g. calcium, cobalt or manganese ions.
  • Cys residue in position 692 of full length Factor VIII is substituted by a Ser residue.
  • a preferred Factor VIII derivative is a calcium-bound complex of the M r 92000 and M r 77/80000 doublet subunits of human Factor VIII wherein Cys 692 has been replaced with Ser.
  • the Factor VIII derivatives are derivatives in which also Glu 720 is deleted or substituted by another amino acid residue selected from the group consisting of Gln, Ser, Thr, Val and Ala.
  • the Factor VIII derivatives are derivatives in which also Tyr 729 is deleted or substituted by another amino acid residue selected from the group consisting of Val, Ala and lie.
  • the derivatives of Factor VIII wherein Glu 720 is deleted or substituted by another amino acid residue and/or wherein Tyr 729 is deleted or substituted by another amino acid residue show higher resistance against cleavage by enzymatic activity present in the medium which gives rise to an increased yield of the desired Factor VIII derivatives.
  • the invention also relates to a method for preparing a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues, comprising culturing a host cell transformed with a gene encoding the
  • Factor VIII derivative under conditions wherein the gene is expressed and the expressed product secreted, and isolating the Factor VIII derivative.
  • the Cys residue in position 692 of full length Factor VIII is substituted by a Ser residue.
  • Factor VIII derivative produced is in the form of a calciumbound complex of the M r 92000 and M r 77/80000 doublet subunits of human Factor VIII wherein Cys 692 has been replaced with Ser.
  • a Factor VIII derivative is produced wherein Glu 720 is deleted or substituted by another amino acid residue selected from the group consisting of Gin, Ser, Thr, Val and Ala.
  • a Factor VIII derivative is produced, wherein Tyr 729 is deleted or substituted by another amino acid residue selected from the group consisting of Val, Ala and lie.
  • the gene encoding the Factor VIII derivatives of the invention may be prepared from DNA encoding the corresponding human Factor VIII by conventional site specific mutagenesis.
  • the DNA may be wholly or partly cDNA, chromosomal DNA and/or synthetic DNA.
  • the method of the invention may be carried out in a manner analogous to those described in the patent applications listed above.
  • the resulting Factor VIII derivative may be purified by standard techniques.
  • the Factor VIII derivative may be produced from Factor VIII isolated from plasma by methods known per se, e.g. as described in EP patent No. 83483, EP patent No. 150735 or EP patent No. 197901 by deleting or substituting Cys 692 with Ser.
  • substitution may be carried out by cleaving off a part of the A2 domain and coupling with a complementary fragment having the desired amino acid sequence.
  • the complementary fragment may be produced by chemical synthesis or substituting the desired Cys residue in a fragment isolated from a plasma source in a manner known per se or produced by recombinant techniques.
  • the coupling may also be carried out by transesterification techniques introducing the desired complementary fragment into the remaining part of the A2 domain possessing a suitable leaving group by a manner known per se.
  • the invention relates to a gene encoding a
  • Such a gene comprises a DNA encoding said human Factor VIII derivative and may comprise cDNA, chromosomal DNA and/or synthetic DNA.
  • the invention relates to a pharmaceutical preparation comprising a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues in admixture with a parenterally acceptable vehicle or excipient.
  • a pharmaceutical preparation according to the invention may comprise further pharmaceutical excipients well known to those skilled in the art. These include, for example, various bulking agents, additional buffering agents, antioxidants, preservatives, stabilizers, and the like.
  • the invention relates to the use of a Factor VIII derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues for the preparation of a pharmaceutical preparation for the treatment of diseases caused by an absence or deficiency of the Factor VIII of a subject.
  • the invention relates to a method for preventing or treating diseases caused by absence or deficiency of Factor VIII:C in a subject comprising administering to the subject a pharmaceutically active amount of a Factor VIII :C derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues in admixture with a pharmaceutically acceptable vehicle or excipient.
  • Such diseases may e.g. be haemophilia A, both patients suffering from lack of Factor VIII due to lack of production or induction of Factor VIII being inactive, and inhibitor patients developing antibodies to Factor VIII.
  • the derivatization itself may modify the epitope of Factor VIII recognized by the antibodies of an inhibitor patient and thus be used directly for treating the haemophilia and bypassing the inhibitor activity without having to take any special measures to neutralize or "by-pass" the antibodies.
  • the Factor VIII derivatives of the invention may show prolonged in vivo activity.
  • the invention also relates to a method of preparing a pharmaceutical preparation comprising a Factor VIII:C derivative comprising a functional A2 domain in which one or more cysteine residues has been deleted or substituted by a one or more other amino acid residues comprising mixing the Factor VIII derivative with pharmaceutically acceptable vehicle and/or excipient and forming a suitable dosis form of the pharmaceutical preparation.
  • a suitable dosis form may e.g. be a lyophilized powder to be reconstituted with water for injection.
  • Such lyophilized powder may be presented in a vial or in a prefilled syringe or pen device, e.g. a dual chamber syringe.
  • the Factor VIII derivatives of the invention may also be presented in the form of a solution to be used in e.g. a pen device.
  • full length Factor VIII designates the full molecule comprising the amino acid residues 1-2332 as disclosed in Nature (1984) 312:339.
  • Factor VIII-HC “heavy chain” or “HC” designates the A1-A2 repeats of Factor VIII comprising the amino acid residues 1-740 of full length Factor VIII as disclosed in Nature (1984) 312 :341.
  • Factor VIII-LC designates the A3-C1-C2 repeats of Factor VIII comprising the amino acid residues 1649-2332 as disclosed in Nature (1984) 312:341.
  • functional A2 domain is used herein to designate a polypeptide having an amino acid sequence only deviating from the amino acid sequence of the A2 domain of human Factor VIII to an extent not having an adverse effect on the overall coagulant activity in terms of specific activity (International Units of Factor VIII activity per mg protein) and duration of the coagulant activity.
  • host cell is used to designate cells which may be employed when preparing the Factor VIII derivatives of the invention by recombinant methods.
  • Such cells are preferably mammalian cells and include for example COS cells, Chinese hamster ovary (CHO) cells, mouse kidney cells, hamster kidney cells, HeLa cells HepG2 cells, or the like.
  • amino acid residue is used in the present specification to designate a naturally occurring ⁇ -amino acid residue different from the amino acid residue present in the native polypeptide.
  • Buffer A 50 mM imidazole, 0.15 mM NaCl, 0.1% BSA, pH 7.4.
  • Chromo ⁇ enic Assay The activity of Factor VIII was measured in a chromogenic assay (Coatest, Chromogenix), as described by the manufacturers, except that all reactions were carried out at room temperature and that the incubation times were altered:
  • Phospholipid, Factor IXa + Factor X, CaCl 2 and the diluted sample was incubated 15 min before adding the substrate + the thrombin inhibitor, and the colour reaction was allowed to take place for 10 min.
  • EXAMPLE 1 Expression of the M r 92000 subunit of human Factor VIII wherein Cys 692 has been replaced with Ser and combination with the M r 80000 subunit of human Factor VIII to form a complex showing coagulant activity.
  • the transfected cells were grown as described by Burke et el, and culture medium was harvested after 24 hours and diluted 6 fold with buffer A, and 50 ⁇ l was incubated with 50 ⁇ l Factor VIII-LC prepared as disclosed in WO 88/00210 diluted to 17 U/ml in buffer A. 20 ⁇ l 1 mM mercaptoethanol and 14 ⁇ l 0.1 M MnCl 2 was added. After 18 hours incubation at 22oC, Factor VIII activity was confirmed by Coatest analysis.
  • C0S7 cells were cotransfected with a plasmid pSVF8-92 encoding the Factor VIII-HC as described in EP 232112 in which the codon encoding Cys in position 692 was replaced by a codon encoding Ser by site mutagenesis as described in Norris et al (ibid) and a plasmid pSVF8-80.
  • the resulting transformed cells were cultured in the same manner as described in WO 91/07490. Media samples were collected after 24 hours expression at 37 oC and assayed for the contents of Factor VIII activity by chromogenic activity assay (Coatest). The results of double transfections are stated in the below Table also showing the results of a sample of a corresponding wild type complex not having the substitution.

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  • Health & Medical Sciences (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un nouveau dérivé du facteur VIII, qui comprend un domaine fonctionnel A2 dans lequel un ou plusieurs résidus de cystéine ont été supprimés ou substitués par un ou plusieurs résidus d'acide aminé. Le dérivé du facteur VIII présente un effet coagulant du facteur VIII qui est stable durant une longue période.
PCT/DK1995/000008 1994-01-07 1995-01-06 Derives du facteur viii WO1995018827A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU13825/95A AU1382595A (en) 1994-01-07 1995-01-06 Factor viii derivatives

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK3294 1994-01-07
DK00032/94 1994-01-07

Publications (1)

Publication Number Publication Date
WO1995018827A1 true WO1995018827A1 (fr) 1995-07-13

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0808901A2 (fr) * 1996-05-24 1997-11-26 Immuno Ag Préparation pharmaceutique avec une activité procoagulante de facteur VIII et une activité de liaison du vWF
US5869292A (en) * 1995-11-13 1999-02-09 Immuno Ag Hybrid proteins with modified activity
WO2000071714A3 (fr) * 1999-05-24 2001-01-18 American Nat Red Cross Procedes de reduction de la clairance du facteur viii et compositions correspondantes
WO2002060951A3 (fr) * 2001-01-12 2003-02-27 American Nat Red Cross Methodes et compositions permettant de reduire la clairance du facteur viii, mediee par l'heparan-sulfate proteoglycane
EP1424344A1 (fr) * 2002-11-29 2004-06-02 Aventis Behring Gesellschaft mit beschränkter Haftung ADNc modifié du facteur VIII et dérivés
EP1454916A1 (fr) * 2002-11-29 2004-09-08 ZLB Behring GmbH ADNc modifié du facteur VIII et dérivés
EP1502921A1 (fr) * 2003-07-29 2005-02-02 ZLB Behring GmbH Mutants du Facteur VIII (FVIII) humain recombinants ayant une meilleure stabilité
US7211559B2 (en) 2003-10-31 2007-05-01 University Of Maryland, Baltimore Factor VIII compositions and methods

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988000210A1 (fr) * 1986-06-24 1988-01-14 Nordisk Gentofte A/S Procede pour produire un complexe actif de coagulation des fragments du facteur viii
EP0294910A1 (fr) * 1987-06-12 1988-12-14 Immuno Ag Protéines à activité de facteur VIII, procédé pour leur production utilisant des cellules modifiées par génie génétique et compositions pharmaceutiques les contenant
WO1992006999A1 (fr) * 1990-10-17 1992-04-30 The Scripps Research Institute Fragments therapeutiques du factor de von willebrand

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988000210A1 (fr) * 1986-06-24 1988-01-14 Nordisk Gentofte A/S Procede pour produire un complexe actif de coagulation des fragments du facteur viii
EP0294910A1 (fr) * 1987-06-12 1988-12-14 Immuno Ag Protéines à activité de facteur VIII, procédé pour leur production utilisant des cellules modifiées par génie génétique et compositions pharmaceutiques les contenant
WO1992006999A1 (fr) * 1990-10-17 1992-04-30 The Scripps Research Institute Fragments therapeutiques du factor de von willebrand

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6156888A (en) * 1995-11-13 2000-12-05 Baxter Aktiengesellschaft Hybrid proteins with modified activity
US6130203A (en) * 1995-11-13 2000-10-10 Baxter Aktiengesellschaft Hybrid proteins with modified activity
US5869292A (en) * 1995-11-13 1999-02-09 Immuno Ag Hybrid proteins with modified activity
US5910481A (en) * 1995-11-13 1999-06-08 Immuno Ag Hybrid proteins with modified activity
US6051418A (en) * 1995-11-13 2000-04-18 Immuno Ag Hybrid proteins with modified activity
EP0808901A2 (fr) * 1996-05-24 1997-11-26 Immuno Ag Préparation pharmaceutique avec une activité procoagulante de facteur VIII et une activité de liaison du vWF
EP0808901A3 (fr) * 1996-05-24 1999-05-06 Immuno Ag Préparation pharmaceutique avec une activité procoagulante de facteur VIII et une activité de liaison du vWF
WO2000071714A3 (fr) * 1999-05-24 2001-01-18 American Nat Red Cross Procedes de reduction de la clairance du facteur viii et compositions correspondantes
WO2002060951A3 (fr) * 2001-01-12 2003-02-27 American Nat Red Cross Methodes et compositions permettant de reduire la clairance du facteur viii, mediee par l'heparan-sulfate proteoglycane
US7615622B2 (en) 2001-01-12 2009-11-10 University Of Maryland, Baltimore Methods and compositions for reducing heparan sulfate proteoglycan-mediated clearance of factor VIII
EP1454916A1 (fr) * 2002-11-29 2004-09-08 ZLB Behring GmbH ADNc modifié du facteur VIII et dérivés
EP1424344A1 (fr) * 2002-11-29 2004-06-02 Aventis Behring Gesellschaft mit beschränkter Haftung ADNc modifié du facteur VIII et dérivés
EP1502921A1 (fr) * 2003-07-29 2005-02-02 ZLB Behring GmbH Mutants du Facteur VIII (FVIII) humain recombinants ayant une meilleure stabilité
US7211559B2 (en) 2003-10-31 2007-05-01 University Of Maryland, Baltimore Factor VIII compositions and methods

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Publication number Publication date
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