WO1995013300A1 - Nouveaux polypeptides du facteur viii - Google Patents

Nouveaux polypeptides du facteur viii Download PDF

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Publication number
WO1995013300A1
WO1995013300A1 PCT/DK1994/000423 DK9400423W WO9513300A1 WO 1995013300 A1 WO1995013300 A1 WO 1995013300A1 DK 9400423 W DK9400423 W DK 9400423W WO 9513300 A1 WO9513300 A1 WO 9513300A1
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factor viii
leu
amino acid
ser
val
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PCT/DK1994/000423
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Marianne Kjalke
Mirella Ezban Rasmussen
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Novo Nordisk A/S
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Priority to AU81400/94A priority Critical patent/AU8140094A/en
Publication of WO1995013300A1 publication Critical patent/WO1995013300A1/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 to new Factor VIII polypeptides showing coagulant activity, a method for the preparation thereof, pharmaceutical preparations comprising the new Factor VIII polypeptides, the use of these polypeptides for the preparation of a pharmaceutical preparation especially for the treatment of diseases caused by an absence or deficiency of the Factor VIII of a subject.
  • Haemophilia A is an X-chromosome-linked inherited disease which afflicts 1-2 males per 10,000. The desease 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 (S.I. 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 as a series of related polypeptides ranging from M r 160000-40000 with predominant species of M r 92000 (the heavy chain) and M r 80000 (the light chain). This complex pattern has made the analysis of the structure of active Factor VIII very difficult. Factor VIII and the related polypeptides have been described by F. Rotblat et al, Biochemistry (1985) 24:4294-4300; G.A. Vehar et al, Nature (1984) 312:337-342; J.J. Toole et al, Nature (1984) 312:342-347; and M.A.
  • 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 (the heavy chain) and A3-C1-C2 (the light chain).
  • 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 A1 and the A2- domains and between the A2 and B domains, and 41 amino acids is cleaved off from the N-terminus of the light chain.
  • 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 and the isolation and purification process which is, moreover, 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.
  • WO 87/07144 discloses the preparation of deletion analogues of Factor VIII lacking 1-1317 amino acid residues from Ser-373 through Arg-1689. However, no examples discloses the preparation of analogues comprising a shortened A2 domain of Factor VIII, and no results are present showing coagulant activity for Factor VIII analogues comprising a shortened A2 domain.
  • the invention relates to a Factor VIII polypeptide comprising a heavy chain lacking a part of the C terminal part of the A2 domain.
  • the invention in a second aspect, relates to a method for the preparation of a Factor VIII polypeptide comprising a heavy chain lacking a part of the C terminal part of the A2 domain.
  • the invention relates to a pharmaceutical preparation comprising a Factor VIII polypeptide comprising a heavy chain where a part of it or all is lacking a part of the C terminal part of the A2 domain.
  • the invention relates to the use of a Factor VIII polypeptide comprising a heavy chain lacking a part of the C terminal part of the A2 domain for the preparation of a pharmaceutical preparation for the prevention or treatment of diseases caused by absence or deficiency of Factor VIII in a subject.
  • the invention relates to a method for preventing or treating diseases caused by absence or deficiency of Factor VIII.
  • the invention relates to a method of preparing a pharmaceutical preparation comprising a Factor VIII polypeptide of the invention.
  • Fig. 1 shows an SDS-PAGE of Factor VIII polypeptides of the invention as compared to Factor VIII polypeptides, containing the complete A2- domain,
  • Fig. 2 shows RP-HPLC profiles of LysC peptides from the heavy chain of Factor VIII polypeptides of the invention as compared to the heavy chain, containing the complete A2-domain,
  • Fig. 3 shows RP-HPLC profiles of AspN peptides from the heavy chain of Factor VIII polypeptides of the invention as compared to the heavy chain of Factor VIII polypeptides containing the complete A2-domain,
  • Fig. 4 shows the inhibition of Factor VIII activity by a monoclonal antibody for peptides of the invention as compared to Factor VIII polypeptides containing the complete A2-domain and plasma Factor VIII,
  • Fig. 5 shows a time-study of thrombin activation of FVIII polypeptides as measured by SDS-PAGE
  • Fig. 6 shows a time-study of thrombin activation of FVIII polypeptides as measured by a clotting assay.
  • the present invention relates to new Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C- terminal part of full length Factor VIII wherein the heavy chain is shorter than the A1-A2 domain of full length Factor VIII.
  • the heavy and light chains are preferably bridged via a metal ion bridge.
  • a bridge is suitably formed via a divalent ion such as Mn 2+ , Ca 2+ or Co + .
  • the bridge is a calcium bridge.
  • the Factor VIII polypeptides of the invention prefererably lacks a part of the C- terminal part of the A2 domain.
  • Preferred Factor VIII polypeptides of the invention comprises a heavy chain comprising the amino acid residues 1-720 or 1 -729 of the heavy chain of full length Factor VIII.
  • the Factor VIII polypeptide of the invention comprising a heavy chain comprising amino acid residues 1-729 of the full length Factor VIII exhibits a coagulant activity of the same level as Factor VIII polypeptide comprising the full heavy chain.
  • the Factor VIII polypeptide of the invention comprising a heavy chain comprising amino acid residues 1-720 of full length Factor VIII exhibits a specific activity as measured in a chromogenic assay of the same level as Factor VIII polypeptides comprising full heavy chain and a specific activity as measured in a clot assay of about 50%.
  • the Factor VIII polypeptides of the invention normally will comprise a light chain having an amino acid sequence corresponding to amino acids 1649-2332 of the C terminal of full length Factor VIII.
  • the Factor VIII polypeptides comprise a light chain having an amino acid sequence corresponding to amino acids 1690-2332 of the C terminus full length Factor VIII.
  • the invention further relates to a method for preparing a Factor VIII polypeptide comprising a heavy chain having an amino acid sequence corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein the heavy chain shorter than the A1-A2 domain of full length Factor VIII wherein a Factor VIII polypeptide comprising the full A1-A2 domain of full length Factor VIII is treated with a protease cleaving off the C-terminal part of the A2 domain.
  • the Factor VIII polypeptides of the invention may be prepared starting from a Factor VIII polypeptide 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 or produced by recombinant techniques, e.g. as described in the patent applications listed above.
  • the Factor VIII polypeptides of the invention are prepared by coexpession of the heavy and light chains of Factor VIII as disclosed in WO91/07490.
  • Such Factor VIII polypeptides lack the B domain of full length Factor VIII and comprise a heavy chain metal ion-bridged to a light chain showing coagulant activity.
  • the Factor VIII polypeptides of the invention may be generated by proteolytic digestion in the medium.
  • the Factor VIII polypeptides of the invention may be purified and isolated by methods known per se for purification and isolation of Factor VIII polypeptides.
  • the invention also relates to a pharmaceutical preparation comprising a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domains of full length Factor VIII in admixture with a parenterally acceptable vehicle or excipient.
  • the invention relates to the use of a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein the heavy chain is shorter than the A1-A2 domains of full length Factor VIII for the preparation of a pharmaceutical preparation.
  • the Factor VIII polypeptides of the invention are used for the preparation of a pharmaceutical preparation for the prevention or treatment of diseases caused by absence or deficiency of Factor VIII in a subject.
  • the invention also relates to a method for preventing or treating diseases caused by absence or deficiency of Factor VIII in a subject comprising administering to the subject a pharmaceutically active amount of a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C-terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domains of full length Factor VIII in admixture with a pharmaceutically acceptable vehicle or excipient.
  • the invention in a further aspect, relates to a method of preparing a pharmaceutical preparation comprising a Factor VIII polypeptide comprising a heavy chain having an amino acid sequences corresponding to the amino acid sequence of the N-terminal part of full length Factor VIII and a light chain having an amino acid sequence corresponding to the amino acid sequence of the C- terminal part of full length Factor VIII wherein all or a part of the Factor VIII has a heavy chain shorter than the A1-A2 domains of full length Factor VIII with pharmaceutically acceptable vehicle and/or excipients and forming a suitable dosis form of the pharmaceutical preparation.
  • full length Factor VIII designates the full molecule comprising the amino acid residues 1-2332 as disclosed in Nature (1984) 312:339.
  • HC heavy chain
  • light chain designates the A3-C1-C2 repeats of Factor VIII comprising the amino acid residues 1649-2332 as disclosed in Nature (1984) 312:341.
  • Recombinant Factor VIII lacking the B domain was purified by a procedure including an affinity chromatography step using a monoclonal antibody directed against the C-terminal part of the heavy chain.
  • a part of the Factor VIII did not bind to the column, due to C-terminal truncation of the A2-domain of the heavy chains.
  • Two Factor VIII forms were purified from the fractions not bound to the antibody column.
  • peptide mapping and isolation of C-terminal peptides by affinity chromatography followed by amino acid sequencing and mass spectro- metry it is shown, that the two C-terminal truncated forms of Factor VIII contains heavy chains. consisting of amino acids 1-720 (FVIII(HC:1-720)) and 1-729
  • FVIII(HC:1-729) Factor VIII bound to the antibody column contains a heavy chain consisting of amino acid 1 -740 corresponding to the entire A1 and A2-domains.
  • FVIII (HC: 1-729) have the same specific activity as FVIII(HC:1-740), and is activated by thrombin at a similar rate.
  • FVI II (HC: 1-720) have the same specific activity when the activity is measured in a chromogenic assay, however, the specific activity is a factor two lower when the specific activity is measured in a clotting assay. Similary, FVIII (HC: 1-720) is activated by thrombin at a slower rate and to a lower level compared with FVIII(HC:1-740), FVIII(HC:1- 729), and plasma Factor VIII.
  • Conditioned medium comprising recombinant Factor VIII in the form of a complex of the M r 90000 and M r 80000 subunits of Factor VIII joined by a calcium bridge prepared as disclosed in WO 91/07490 containing 20 U Factor VIII pr. ml was filtrated.
  • the filtrate was applied to a cation-exchange S-F (Pharmacia LKB) column and eluted using a salt gradient (increasing the ionic strength).
  • the eluate from the column was loaded on an immunoaffinity column consisting of an antibody (F25-lgG) directed against the C-terminal part of the heavy chain coupled to CNBr activated Sepharose 4B (Pharmacia) equilibrated with 50 mM TrisCI pH 7.3 containing 150 mM NaCI, 10 mM CaCI 2 , 10% (v/v) glycerol and 0.02% (v/v) Tween 80 at room temperature.
  • the column was washed with 6 volumes of starting buffer.
  • the flow-through containing FVIII (HC: 1-729) and FVIII (HC: 1-720) was collected.
  • the column was washed further with 4 volumes 50 mM TrisCI pH 7.3 containing 0.65 M NaCI before eluting with 2.5 volumes of 20 mM TrisCI pH 7.3 containing 2.5 M NaCI, 50% (v/v) ethylenglycol, 10 mM CaCI 2 and 0.02% Tween 80.
  • the eluate containing FVIII(HC: 1-740) were desalted on a Sephadex G25 column (5.3 x 32 cm, Pharmacia).
  • the F25-lgG was prepared by purifying Factor VIII HC from plasma as described in WO 88/00210. Using this isolated Factor VIII HC the monoclonal antibody (F25- IgG) was prepared using the procedure disclosed in Thromb. Haemostas 1985:54, 586-590.
  • the Factor VIII forms were finally purified on a MonoQ PC 1.6/5 column using the SMART system (Pharmacia). Approximately 800 U FVIII(HC: 1-740) or FVIII(HC:1- 729) and FVIII(HC: 1-720) was loaded on the column equilibrated with 20 mM TrisCI pH 7.5 containng 150 mM NaCI, 10 mM CaCI 2 , 10% glycerol (v/v) and 0.02% (v/v) Tween 80 at room temperature at a flow of 100 ⁇ l min '1 . After washing, a 30 min gradient of 150 to 500 mM NaCI in the buffer was applied.
  • AspN endoprotease digestions were made on heavy chains from FVIII(HC: 1-740) (approximately 1.6 nmol), FVIII (HC: 1-729) (approximately 0.9 nmol), and FVIII- (HC: 1-720) (approximately 0.2 nmol).
  • FVIII- HC: 1-720
  • redissolvation in 8 M urea 0.75 ⁇ g AspN endoprotease (Boehringer Mannheim) in 10 mM TrisCI pH 7.5 was added to each sample to a final concentration of urea of 1 M. After 1 hour at 22°C another 0.75 ⁇ g AspN endoprotease was added to each sample and the reactions were continued for 15 hours before stopping by adding 10% trifluoro- acetic acid to pH 2.
  • FVIII(HC:1-740), FVIII(HC: 1 -729), and FVIII (HC: 1-720) were diluted into 1.25 ml of 20 mM TrisCI pH 7.5 containing 150 mM NaCI, 10 mM CaCI 2 , 10% (v/v) glycerol and 0.02% (v/v) Tween 80 to a final concentration of 20 U/ml.
  • the samples were preincubated 2 min at 37°C before a 100 ⁇ l sample was withdrawn an added to 20 ⁇ l icecold 50% (w/w) trichloracetic acid containing 0.2% (w/w) sodiumdeoxycolat. Human ⁇ -thrombin (Boehringer Mannheim) was added to the remaining Factor VIII to a final concentration of 0.1 U/ml, and 100 ⁇ l samples were withdrawn at the times indicated in Fig. 5 and the cleavage stopped as described above. The samples were incubated 30 min at 4°C before centrifugation 10 min at 18000 x g, and the precipitate analyzed by SDS-PAGE.
  • the Factor VIII concen ⁇ tration was 0.9 U/ml, and the final volume 0.8 ml. Samples of 50 ⁇ l were withdrawn at times indicated on Fig. 6 and analyzed by a clotting assay on Amelung Coagulometer as described below.
  • LysC endoprotease peptides from FVIII(HC:1-720) heavy chain was added to 1 ml anhydrotrypsin agarose (Clontec) packed in a BioRad column with a diametre of 0.9 cm equilibrated with 50 mM sodium acetate pH 5.0 containing 20 mM CaCI 2 at a flow of 0.18 ml min "1 .
  • the column was washed with 20 volumes of starting buffer and fractions of 0.5 ml collected. Absorbance at 227 nm was detected.
  • the bound peptides were eluted with 10 volumes 5 mM HCI pH 2.5.
  • the pH of the pooled fractions containing the non-bounded peptides was adjusted to pH 2 by adding 10% trifluoroacetic acid before rechromatography on reverse phase-HPLC. A sample containing only LysC endoprotease and buffer was runned in parallel.
  • the peptides (10-100 pmol) were sequenced on an Applied Biosystem model 477A sequencer equipped with on-line model 120A HPLC using standard pro ⁇ grams as described by the manufacturer. Mass spectra were recorded with a time-of-flight plasma desorption mass spectrometre (Bio-Ion 20, Applied Biosystem) at 16 kV acceleration.
  • Samples for amino acid analyses were hydrolyzed for 20 hours under vacuum at 110°C in 6 M HCI containing 0.1% phenol and 0.1 % dithiodipropionic acid (Barkholt and Jensen (1989) Anal. Biochem. 177: 318-322). For determination of specific activity 2-3 hydrolyses of each sample were made. The samples were acetone precipitated and redissolved in H 2 0 to approximately 0.1 ⁇ g ⁇ l '1 . Norleu- sine was added to each sample (1.5 nmol each) as internal standard. The samples were dryed in a speed-vac, and hydrolyses performed as decsribed above.
  • Factor VIII The activity of Factor VIII was measured in a chromogenic assay (Coatest, Cro- mogenix), 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, CaCI 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.
  • Clotting activity of Factor VIII was measured as the ability to restore clotting activity of FVIII deficient plasma (ACL analyses, IL Laboratories, or Amelung coagulometer, Pharmacia). Clotting time analysis on the ACL instrument was carried out as described by the manufacturer. All reagens were from IL Laboratories except Factor VIII deficient plasma and APTT reagens that were from Organon Teknika.
  • SDS-PAGE was performed on reduced samples in 7.5% polyacrylamide gels as described in Biochemistry (1991) 30:1533-1537 using the BioRad Mini-Protean system. The gels were silver stained.
  • FIG. 1 shows SDS-PAGE of the three Factor VIII forms and thrombin generated fragments from theese compared with plasma Factor VIII containing no B-domain.
  • FVIII(HC: 1-740) have similar Mr as the plasma Factor VIII, while FVIII(HC: 1-729) and FVIII(HC: 1-720) had slightly lower Mr of the heavy chains. The Mr differences is located in the A2-domains as seen by the Mr of the fragments generated by thrombin cleavage (see arrows on Fig. 1).
  • Fig. 2 shows the LysC peptide maps of reduced and alkylated heavy chain from the recombinant Factor VIII forms. Peaks not seen in all three maps were analyzed by amino acid sequencing and mass spectrometry (see Table I below).
  • FVIII(HC: 1-740) A 734-740 NNAIEPR 814.4 813.9 FVIII(HC: 1-740) B 714-733 NTGDYYEDSY- ND 2347.4 EDISaYLLSK 214-230 NSLMQDRDAA- ND 1918.1 SARAWPK
  • the peptide marked A corresponding to amino acid 734-740, was only present in FVIII(HC:1-740), showing that only this form contains the full-length A2-domain.
  • FVIII(HC: 1-729) contained the peak marked D, which corresponds to amino acid 714-729. Because LysC is not cleaving peptides C-terminal to Tyr, this indi ⁇ cates that FVIII(HC:1-729) have C-terminus at Tyr729.
  • Both FVIII(HC:1-729) and FVIII(HC: 1-720) contains the peaks marked E corresponding to amino acid 714- 720. This peak is not seen in FVIII(HC: 1-740) indicating that some Factor VIII have C-terminus at Glu720.
  • FVIII- (HC: 1-740) contains the peaks marked B and C both containing amino acid 714- 733.
  • the prescence of this peptide in two peaks could be due to partial sulfata- tion of one or more of the expected sulfate groups at Tyr 118, Tyr 119 and 723 as seen in the full-length FVIII molecule expressed in Chinese Hamster ovary cells (Mikkelsen et al., Biochemistry (1991), 30, 1533-1537.
  • AspN peptide mapping of the unreduced heavy chains from FVIII(HC: 1-740), FVIII (HC: 1-729), and FVIII(HC: 1-720) is shown in Fig. 3.
  • Table II shows amino acid sequences and mass spectrometry data of the peaks deviating in retention time among the three maps.
  • the peak marked A in the map of FVIII(HC: 1-740) corresponds to the C-terminal AspN peptide. Sequencing of the "shoulder" (retention time 27.5 min, Fig. 3) of the peak from FVIII (HC: 1-729) eluting just before peptide A, did not show the se ⁇ quence of the C-terminal peptide (not shown). In accordance with the results of the LysC peptide maps, this shows that only FVIII(HC: 1-740) contains the full- length A2-domain.
  • the C-terminal LysC peptides from the heavy chain of FVIII(HC:1-720) were purifi- ed by anhydrotrypsin affinity chromatography.
  • Anhydrotrypsin is a catalytical inac ⁇ tive derivative of trypsin with the ability of binding peptides with C-terminal Lys or Arg (Ishii and Kumazaki, (1988) in Methods in Protein Seguence Analysis. (B. Witman-Liebold, ed.) pp. 156-163, Springer Verlag, Berlin).
  • FVIII(HC:1-740), FVIII(HC: 1-729), and FVIII (HC: 1-720) was measured by both a chromogenic assay and a one-stage clotting assay (see Table IV, below).
  • the specific activity of all the Factor VIII forms was approximately 1.0 x 10 4 U/mg as determined by the chromogenic assay.
  • FVIII (HC: 1-740) and FVIII (HC: 1-729) also have the same specific activity within the experimental error as determined by the clotting assay.
  • FVIII (HC: 1-720) have a specific activity a factor two lower as determined in the clotting assay.
  • the monoclonal antibody (F25-lgG) used for separation of FVIII(HC: 1-740) from FVIII(HC:1-729) and FVIII(HC: 1-720) was used for inhibition of activity of plasma Factor VIII and the three recombinant Factor VIII forms as measured by a chromogenic assay and a one-stage clotting assay (Fig. 4).
  • the antibody inhibits the activity of FVIII(HC: 1-740) and plasma Factor VIII at a similar degree as measured in the clotting assay, but was not able to inhibit in the chromogenic assay. In the clotting assay the time of clotting of Factor VIII deficient plasma is measured.
  • the Factor VIII form is incubated with Factor IXa, Factor X and phospholipid for 15 minutes before the Factor Xa substrate assay is added. This means, that any differences in affinity of for example thrombin to Factor VIII would be masked by the long incubation time in the chromogenic assay but not in the clotting assay. As expected, the inhibitory effect of the antibody was not seen for FVIII(HC:1-729) and FVIII(HC:1-720).
  • Plasma Factor VIII, FVIII(HC:1-740) and FVIII(HC:1-729) is activated at similar rates, while FVIII(HC: 1-720) is activated more slowly.
  • SDS-PAGE Fig. 5
  • Ser Pro lie Thr Phe Leu Thr Ala Gin Thr Leu Leu Asp Leu Gly 290 295 300

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Abstract

Un polypeptide du facteur VIII comprend une chaîne lourde, dotée d'une séquence d'acides aminés correspondant à celle du terminal N du facteur VIII complet, et une chaîne légère, dotée d'une séquence d'acides aminés correspondant à celle du terminal C du facteur VIII complet, la chaîne lourde étant plus courte que le domaine A1-A2 du facteur VIII complet. Ce polypeptide donne l'effet de coagulation du facteur VIII et peut s'utiliser pour prévenir ou traiter des maladies causées par l'absence ou une déficience du facteur VIII chez un sujet.
PCT/DK1994/000423 1993-11-12 1994-11-10 Nouveaux polypeptides du facteur viii WO1995013300A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001012836A1 (fr) * 1999-08-13 2001-02-22 Fred Hutchinson Cancer Research Center Cristal de produit de recombinaison proteique tronque renfermant un domaine c2 du facteur de coagulation viii, avec ou sans ligand lie, et procedes d'utilisation
WO2001019992A2 (fr) * 1999-09-14 2001-03-22 Baxter Aktiengesellschaft Anticorps d'activation du facteur ix/du facteur ixa et derives d'anticorps
US6376463B1 (en) 1992-04-07 2002-04-23 Emory University Modified factor VIII
US7297336B2 (en) 2003-09-12 2007-11-20 Baxter International Inc. Factor IXa specific antibodies displaying factor VIIIa like activity
EP2258860A1 (fr) 2005-03-29 2010-12-08 Octapharma AG Procédé pour l'isolation de protéines recombinantes
WO2011095604A1 (fr) 2010-02-04 2011-08-11 Octapharma Biopharmaceuticals Gmbh Prolongation de la demi-vie de protéines
US8871439B2 (en) 2005-06-30 2014-10-28 Octapharma Ag Serum-free stable transfection and production of recombinant human proteins in human cell lines

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WO1987007144A1 (fr) * 1986-05-29 1987-12-03 Genetics Institute, Inc. Nouvelles proteines stimulant la coagulation du sang
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

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Publication number Priority date Publication date Assignee Title
WO1987007144A1 (fr) * 1986-05-29 1987-12-03 Genetics Institute, Inc. Nouvelles proteines stimulant la coagulation du sang
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

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Title
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6376463B1 (en) 1992-04-07 2002-04-23 Emory University Modified factor VIII
US7033791B2 (en) 1992-04-07 2006-04-25 Emory University Nucleic acid molecules encoding modified factor VIII proteins, expression products, and methods of making the same
WO2001012836A1 (fr) * 1999-08-13 2001-02-22 Fred Hutchinson Cancer Research Center Cristal de produit de recombinaison proteique tronque renfermant un domaine c2 du facteur de coagulation viii, avec ou sans ligand lie, et procedes d'utilisation
WO2001019992A2 (fr) * 1999-09-14 2001-03-22 Baxter Aktiengesellschaft Anticorps d'activation du facteur ix/du facteur ixa et derives d'anticorps
WO2001019992A3 (fr) * 1999-09-14 2001-09-27 Baxter Ag Anticorps d'activation du facteur ix/du facteur ixa et derives d'anticorps
US7033590B1 (en) 1999-09-14 2006-04-25 Baxter Aktiengesellschaft Factor IX/factor IXa activating antibodies and antibody derivatives
US7279161B2 (en) 1999-09-14 2007-10-09 Baxter Aktiengesellschaft Factor IX/factor IXa activating antibodies and antibody derivatives
US7297336B2 (en) 2003-09-12 2007-11-20 Baxter International Inc. Factor IXa specific antibodies displaying factor VIIIa like activity
EP2258860A1 (fr) 2005-03-29 2010-12-08 Octapharma AG Procédé pour l'isolation de protéines recombinantes
US9388402B2 (en) 2005-03-29 2016-07-12 Octapharma Ag Method for improved isolation of recombinantly produced proteins
EP3467116A1 (fr) 2005-03-29 2019-04-10 Octapharma AG Procédé d'isolement de protéines produites par recombinaison
US10626431B2 (en) 2005-03-29 2020-04-21 Octapharma Ag Method for improved isolation of recombinantly produced proteins
US8871439B2 (en) 2005-06-30 2014-10-28 Octapharma Ag Serum-free stable transfection and production of recombinant human proteins in human cell lines
US9273325B2 (en) 2005-06-30 2016-03-01 Carola Schroeder Serum-free stable transfection and production of recombinant human proteins in human cell lines
US9512457B2 (en) 2005-06-30 2016-12-06 Octapharma Ag Serum-free stable transfection and production of recombinant human proteins in human cell lines
US9796986B2 (en) 2005-06-30 2017-10-24 Octapharma Ag Serum-free stable transfection and production of recombinant human proteins in human cell lines
WO2011095604A1 (fr) 2010-02-04 2011-08-11 Octapharma Biopharmaceuticals Gmbh Prolongation de la demi-vie de protéines

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AU8140094A (en) 1995-05-29

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