EP0253455A1
Expired / Lapsed
EP
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Method for the preparation of proteins with factor VIII activity by microbial host cells; expression vectors, host cells, antibodies
Assignee: Gist Brocades NV
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Filing: 1987-07-20
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Granted: —
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Expires: 2007-07-20
Abstract
Abstract Microbial systems are provided for producing peptides having biological and physiological factor VIII activity, in being immunologically cross-reactive with factor VIII and physiologically active in providing for clotting activity. The protein products wary from immunologically cross-reactive fragments to proteins comprising the two Factor VIII subunits joined together directly or by a bridge. High yields of the products are obtained in isolatable and biologically active form.
Inventors
- 👤 Albert Johannes Joseph Van Ooyen
- 👤 Peter Michael Andreoli
- 👤 Jan Aart Van Mourik
- 👤 Hans Pannekoek
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-08 11:30
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/75; A; A61; A61P; A61P7/00; A61P7/02; C07; C07K; C07K14/00; C07K14/435; C07K14/745; C07K14/755; C07K16/00; C07K16/18; C07K16/36; C12N15/11; C12N15/62; C12N15/625; C12N15/78; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/86; A61K; A61K38/00; C07K2319/00; C07K2319/01; C07K2319/036; G01N2333/00; G01N2333/435; G01N2333/745; G01N2333/755
Claims Analysis
Total Claims: 48
Claim 1
Independent Claim
52 words
An expression cassette comprising in the direction of transcription, a transcriptional and translational initiation regulatory region, optionally a secretion signal sequence, an open reading frame encoding an amino acid sequence immunologically cross-reactive with factor VIII, and a translational and transcriptional termination regulatory region, said regulatory regions being functional in a microbial host.
Claim 2
Dependent Claim
52 words
An expression cassette comprising in the direction of transcription, a transcriptional and translational initiation regulatory region, optionally a secretion signal sequence, an open reading frame encoding an amino acid sequence immunologically cross-reactive with factor VIII, and a translational and transcriptional termination regulatory region, said regulatory regions being functional in a microbial host.
Claim 3
Dependent Claim
30 words
An expression cassette according to claim 1, wherein said open reading frame substantially encodes at least one of the 80 kDA protein or the 92 kDa protein of factor, VIII.
Claim 4
Dependent Claim
32 words
An expression cassette according to claim 2, wherein said open reading frame encodes the 80 kDA and 92 kDa proteins joined by other means than the natural polypeptide bridge of factor VIII.
Claim 5
Dependent Claim
64 words
A vector comprising a replication system functional in a microbial host and an expression cassette comprising in the direction of transcription, a transcriptional and translational initiation regulatory region, optionally a secretion signal sequence, an open reading frame encoding an amino acid sequence immunologically cross-reactive with factor VIII, and a translational and transcriptional termination regulatory region, said regulatory regions being functional in a microbial host.
Claim 6
Dependent Claim
64 words
A vector comprising a replication system functional in a microbial host and an expression cassette comprising in the direction of transcription, a transcriptional and translational initiation regulatory region, optionally a secretion signal sequence, an open reading frame encoding an amino acid sequence immunologically cross-reactive with factor VIII, and a translational and transcriptional termination regulatory region, said regulatory regions being functional in a microbial host.
Claim 7
Dependent Claim
19 words
A vector according to claim 4, wherein said vector further comprises a marker for selection in a microbial host.
Claim 8
Dependent Claim
29 words
A vector according to claim 4, wherein said open reading frame substantially encodes at least one of the 80 kDa protein or the 92 kDa protein of factor VIII.
Claim 9
Dependent Claim
17 words
A vector according to claim 4, wherein said open reading frame substantially encodes the 80 kDA protein.
Claim 10
Dependent Claim
17 words
A vector according to claim 4, wherein said open reading frame substantially encodes the 92 kDa protein.
Claim 11
Dependent Claim
31 words
A vector according to claim 4, wherein said open reading frame encodes the 80 kDA and 92 kDa proteins joined by other means than the natural polypeptide bridge of factor VIII.
Claim 12
Dependent Claim
22 words
A vector according to claim 9, wherein said 80 kDA and 92 kDa proteins are in the natural order of factor VIII.
Claim 13
Dependent Claim
2 words
Plasmid pGB850
Claim 14
Dependent Claim
2 words
Plasmid pGB850
Claim 15
Dependent Claim
2 words
Plasmid pGB852
Claim 16
Dependent Claim
2 words
Plasmid pGB852
Claim 17
Dependent Claim
2 words
Plasmid pGB853
Claim 18
Dependent Claim
2 words
Plasmid pGB853
Claim 19
Dependent Claim
2 words
Plasmid pGB861
Claim 20
Dependent Claim
2 words
Plasmid pGB861
Claim 21
Dependent Claim
2 words
Plasmid pGB864
Claim 22
Dependent Claim
2 words
Plasmid pGB864
Claim 23
Dependent Claim
2 words
Plasmid pOL92
Claim 24
Dependent Claim
2 words
Plasmid pOL92
Claim 25
Dependent Claim
2 words
Plasmid pOL80
Claim 26
Dependent Claim
2 words
Plasmid pOL80
Claim 27
Dependent Claim
2 words
Plasmid pPROM92/80PB
Claim 28
Dependent Claim
2 words
Plasmid pPROM92/80PB
Claim 29
Dependent Claim
3 words
19 Vector pOL5
Claim 30
Dependent Claim
3 words
19 Vector pOL5
Claim 31
Dependent Claim
37 words
The preparation of a microbial host, characterized in that a Bacillus subtilis or a Bacillus licheniformis, which is alpha-amylase deficient and/or exoprotease deficient is transformed with a vector according to any one of claims 4-10 or 16-18.
Claim 32
Dependent Claim
15 words
A transformed microbial host comprising an expression cassette according to any one of claims 1-3.
Claim 33
Dependent Claim
16 words
A microbial host according to claim 21, wherein said host is a bacterium or a yeast.
Claim 34
Dependent Claim
19 words
A microbial host according to claim 22, wherein said bacterium is a Bacillus and the yeast is a Kluyveromyces.
Claim 35
Dependent Claim
16 words
A microbial host according to claim 23, wherein said bacterium is Bacillus subtilis or Bacillus licheniformis.
Claim 36
Dependent Claim
24 words
A Bacillus host according to any one of claims 23-24 wherein said expression cassette is present on the plasmid pOL92 , pOL80 or pPROM92/80PB.
Claim 37
Dependent Claim
13 words
A microbial host according to claim 23, wherein said Kluyvercmyces is Kluyveromyces lactis.
Claim 38
Dependent Claim
24 words
A Kluyveromyces host according to claim 23 or 26, wherein said expression cassette is present on the plasmid pGB850, pGB852, pGB853, pGB861 or pGB864.
Claim 39
Dependent Claim
38 words
A process for producing a polypeptide in isolatable form cross-reactive with factor VIII, said process comprising; cultivating a microbial host according to any of claims 21-27 in a nutrient medium to produce said polypeptide; and isolating said polypeptide.
Claim 40
Dependent Claim
42 words
A process according to claim 28, wherein said open reading frame is joined at its 5' terminus to a DNA sequence encoding a signal sequence functional in said microbial host for secretion of said polypeptide and wherein said signal sequence is processed.
Claim 41
Dependent Claim
13 words
A process according to claim 29, wherein said microbial host is a Kluyveromyces.
Claim 42
Dependent Claim
13 words
A process according to claim 29, wherein said microbial host is a Bacillus.
Claim 43
Dependent Claim
44 words
A pharmaceutical composition comprising a polypeptide produced by the process comprising: cultivating a microbial host according to any of claims 21-27 in a nutrient medium to produce said polypeptide; and isolating said polypeptide, and a pharmaceutically acceptable carrier or diluent and optionally an adjuvant.
Claim 44
Dependent Claim
29 words
A polypeptide produced by the process comprising: cultivating a microbial host according to any of claims 21-27 in a nutrient medium to produce said polypeptide, and isolating said polypeptide.
Claim 45
Dependent Claim
16 words
An antibody to a protein obtained by a process according to any one of claims 28-31.
Claim 46
Dependent Claim
7 words
A monoclonal antibody according to claim 34.
Claim 47
Dependent Claim
57 words
A method of purifying protein having factor VIII activity which comprises bringing a liquid phase comprising protein and a content of impurities into contact with a solid phase comprising antibody according to claim 34 or 35, separating the liquid phase from the solid phase and eluting from the solid phase protein with a reduced content of impurities.
Claim 48
Dependent Claim
55 words
A method of diagnosing a blood sample for factor VIII abnormalities which comprises bringing the sample into contact with an antibody according to claim 34 or 35 and assaying for the presence or absence of a conjugate of the antibody with factor VIII antigen as a measure of the factor VIII protein in the sample.
References Cited (Backward Citations)
EP0150735A2
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EP0160457A1
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