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1. (WO2000063403) RECOMBINANT PROTEIN PRODUCTION IN A HUMAN CELL USING SEQUENCES ENCODING ADENOVIRUS E1 PROTEIN
Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

CLAIMS

1. A method for producing at least one proteinaceous
substance in a cell, comprising providing a eukaryotic cell having a sequence encoding at least one El protein of an adenovirus or a functional homologue, fragment and/or derivative thereof in its genome, which cell does not encode a structural adenoviral protein from its genome or a sequence integrated therein, said method comprising providing said cell with a gene encoding a recombinant proteinaceous substance, culturing said cell in a suitable medium and harvesting at least one proteinaceous substance from said cell and/or said medium.
2. A method for enhancing production of a recombinant proteinaceous substance in a eukaryotic cell, comprising providing said eukaryotic cell with a nucleic acid encoding at least part of said proteinaceous substance, whereby said coding sequence is under control of a CMV-promoter, an EIA promoter or a functional homologue, derivative and/or
fragment of either and further providing said cell with E1A-activity or ElA-like activity.
3. A method according to claim 1 or claim 2, wherein said cell is a mammalian cell .
4. A method to anyone of claims 1-3, wherein said cell is a human cell .
5. A method according to anyone of claims 1-4, wherein at least one of said at least one harvested proteinaceous substance is encoded by said gene.
6. A method for producing at least one human recombinant protein m a cell, comprising providing a human cell having a sequence encoding at least one Ei protein of an adenovirus or a functional derivative, homologue or fragment thereof in its genome which cell does not produce structural adenoviral proteins, with a gene encoding said human recombinant
protein, culturing said cell m a suitable medium and harvesting at least one human recombinant protein from said cell and/or said medium.
7. A method according to anyone of the aforegoing claims, wherein said at least one El protein of adenovirus comprises an EIA protein or a functional homologue, fragment and/or derivative thereof.
8. A method according to anyone of the aforegoing claims, wherein said at least one El protein of adenovirus comprises an EIB protein or a functional homologue, fragment and/or derivative thereof.
9. A method according to anyone of claims 1-8, wherein said cell is capable of producing 2 to 200 -fold more recombinant protein and/or proteinaceous substance than conventional mammalian cell lines.
10. A method according to claim 9, wherein said conventional mammalian cell lines are selected from the group consisting of CHO, COS, Vero, Hela, BHK and Sp-2 cell lines.
11. A method according to any one of the aforegoing claims wherein said recombinant protein and/or proteinaceous substance is a protein that undergoes post-translational and/or peri-translational modifications.
12. A method according to claim 11, wherein said
modifications comprise glycosylation.
13. A method according to any one of claims 1-12, wherein said recombinant protein and/or proteinaceous substance is erythropoietm, or a functional derivative, homologue or fragment thereof .
14. A method according to claim 13, wherein said human cell is capable of producing m excess of 100 units erythropoiet or functional derivatives thereof per million cells m 24 nours
15. A method according to claim 13, wherein said human cell is capable of producing m excess of 500 units erythropoietir or functional derivatives thereof per million cells m 24 nours.
16. A method accordmg to claim 13, wherein said human cell is capable of producing in excess of 1000 units
erythropoietm or functional derivatives thereof per million cells m 24 hours.
17. A method according to claim 13, wherein said human cell is capable of producing in excess of 5000 units
erythropoietm or functional derivatives thereof per million cells m 24 hours.
18. A recombinant mammalian cell immortalized by the presence of at least one adenoviral El protein or a functional
derivative, homologue and/or fragment thereof, and further comprising a nucleic acid in a functional format for
expressing at least one variable domain of an immunoglobulin or a functional derivative, homologue and/or fragment
thereof .
19. A recombinant mammalian cell according to claim 18, wherein said at least one adenoviral El protein comprises an EIA protein or a functional homologue, fragment and/or derivative thereof.
20. A recombinant mammalian cell according to claim 18 or 19, wherein said at least one adenoviral El protein comprises an

EIB protein or a functional homologue, fragment and/or derivative thereof.
21. A recombinant mammalian cell according to any one of claims 18-20, comprising a nucleic acid derived from an adenovirus encoding said at least one adenoviral El protein.

22. A recombinant mammalian cell accordmg to claim 21, wherein said nucleic acid derived from an adenovirus encodes an EIA and/or an EIB protein.
23. A recombinant mammalian cell according to any one of claims 18-22, whereby said cell is derived from a primary cell.
24. A recombinant mammalian cell according to any one of claims 18-23, which is derived from a human cell.
25. A recomDmant mammalian cell according to claim 24, whicn is PER.C6 as deposited under ECACC no. 96022940 or a
derivative tnereof .

26. A recombinant mammalian cell according to any one of claims 18-25, whereby said cell further comprises a nucleic acid encoding E2A or a functional homologue, fragment and/or derivative thereof.
27. A recombinant mammalian cell according to claim 26, wherein said nucleic acid encoding E2A comprises a
temperature sensitive mutant E2A.
28. A recombinant mammalian cell according to any one claims 18-27, wherein said nucleic acid m a functional format for expressing at least one variable domain, encodes a heavy chain, a variable heavy chain, a light chain and/or a variable light chain of an immunoglobulin.
29. A recombinant mammalian cell according to any one of claims 18-28, further comprising another nucleic acid m functional format for expressing at least one counterpart of said at least one variable domain.
30. A recombinant mammalian cell according to anyone of claims 18-29, wherein said nucleic acid m functional format for expressing at least one variable domain and/or at least one counterpart thereof encodes an ScFv.
31. A recombinant mammalian cell according to any one of claims 18-30, wherein at least one of said variable domains comprises a human or humanized ammo acid sequence.
32. A recombinant mammalian cell according to anyone of claim 18-31, wherein at least one of said variable domains is encoded by a nucleic acid under the control of an inducible promoter.
33. A method for producing at least one variable domain of an immunoglobulin comprising culturing a recombinant mammalian cell according to any one of claims 18-32, m a suitable medium and harvesting said at least one variable domain of an immunoglobulin from said recombinant mammalian cell and/or said medium.
34. A method according to claim 33, wherein said recombinant mammalian cell is capable of producing m excess of 10 μg of said at least one variable domain of an immunoglobulin per IO6 cells per day.
35. Use of a recombinant mammalian cell according to claims 18-34, for producing at least one variable domain of an immunoglobulin, said at least one variable domain of an immunoglobulin having post-translational modifications different than that of their isolated natural counterparts.

36. Use according to claim 35, wherein said cell is capable of producing said at least one variable domain of an
immunoglobulin, in excess of 10 μg per 10s cells per day.
37. An immunoglobulin or a functional part, homologue and/or derivative thereof, obtainable by a method according to claim 33 or 34 or by a use according to claim 35 or 36.
38. A pharmaceutical composition comprising at least one variable domain of an immunoglobulin according to claim 37. 39. Use of an immunoglobulin or a functional part, homologue and/or derivative thereof according to claim 37 for the therapeutic treatment of an individual.
40. A method accordmg to anyone of claims 1-12, wherein said recombinant protein and/or said proteinaceous substance comprises a viral protein other than an adenoviral protein.

41. A method accordmg to claim 40, wherein said viral protein comprises at least an influenza virus neuramidase and/or a haemagglutmm.
42. A method according to claim 40, wherein said viral protein comprises an enterovirus protein or a functional equivalent thereof
43. A method according to claim 42, wherein said enterovirus protein comprises a rhmovirus, aphtovirus, or
poliomyelitisvirus protein.
44. A method according to claim 40, wherein said viral protein comprises a herpesvirus protein or a functional equivalent thereof.
45. A method accordmg to claim 44, wherein said herpesvirus protein comprises a herpes symplex virus, pseudorabies virus or bovine herpes virus protein
46. A method according to claim 40, wherein said virus protein comprises an orthomyxovirus protein.
47. A method accordmg to claim 46, wherein said
orthomyxovirus protein comprises an influenza virus, a paramyxovirus, such as newcastle disease virus, a respiratory syncitio virus, a mumps virus or a measles virus protein.

48. A method according to claim 40, wherein said virus protein comprises a retrovirus, a parvovirus or a popavovirus protein.
49. A method according to claim 48, wherein said retrovirus protein comprises a human immunodeficiency virus protein.

50. A method according to claim 40, wherein said virus protein comprises a rotavirus or a coronavirus protein.
51. A method according to claim 50, wherein said rotavirus or coronavirus protein comprises a transmissable
gastroenteπtisvirus or a flavivirus, such as tick-borne encephalitis virus or yellow fever virus protein.
52. A method according to claim 40, wherein said virus protein comprises a togavirus protein, such as rubella virus protein or an eastern-, western-, or venezuelean equine encephalomyelitis virus protein.
53. A method according to claim 40, wherein said virus protein comprises a hepatitis causing virus protein, such as a hepatitis A protein or a hepatitis B virus protein
54. A method according to claim 40, wherein said virus protein comprises a pestivirus protein, such as hog cholera virus protein or a rhabdovirus protein, such as a rabies virus protein.
55. Use of a human cell having a sequence encoding at least one El protein of an adenovirus or a functional derivative, homologue or fragment thereof m its genome which cell does not produce a structural adenoviral protein, for the
production of at least one viral protein for use m a
vaccine .
56. A metnod according to any one of claims 1-17, 33, 34, 40-54, wherein said cell is derived from a primary cell.
57. A method according to any one of claims 1-17, 33, 34, 40- 54 or 56, wherein said human cell is immortalized by the presence of said El encoding sequence.
58. A method according to any one of claims 1-17, 33, 34, 40- 54, 56 or 57, wherein said cell further comprises a sequence encoding E2A or a functional derivative or analogue or fragment thereof in its genome.
59. A method according to claim 58, wherein said E2A encoding sequence encodes a temperature sensitive mutant E2A.
60. A method according to any of claims any one of claims 1-17, 33, 34, 40-54, 56-59, whereby said human cell comprises no other adenoviral sequences .
61. A method according to any one of claims 1-17, 33, 34, 40-54, 56-60, wherein said human cell is capable of growing m suspension.
62. A method according to any one of claims 1-17, 33, 34, 40-54, 56-61, wherein said cell is PER . C6 as deposited under ECACC no. 96022940 or a derivative thereof.
63. A method according to any one of claims 1-17, 33, 34, 40-54, 56-62, wherein said human cell can be cultured m the absence of serum.
64. Use of a human cell having a sequence encoding at least one El protein of an adenovirus or a functional derivative, homologue or fragment thereof m its genome which cell does not produce structural adenoviral proteins for the production of a recombinant protein.
65. Use according to claim 64, whereby said human cell is capable of producing 2-200 fold more recombinant protein than conventional mammalian cell lines.
66. Use according to claim 64 or 65, whereby said cell is derived from a primary cell .
67. Use according to claims 64-66, whereby said human cell is a PER.C6 cell or a derivative thereof.
68. Use according to claims 64-67, wherein said cell further comprises a sequence encoding E2A or a functional derivative or analogue or fragment thereof m its genome.
69. Use according to claim 68, wherein said E2A encoding sequence encodes a temperature sensitive mutant E2A.
70. A recombinant protein obtainable by a method according to any one of claims 1-17, 33, 34, 40-54, 56-63, said
recombinant protein having a human glycosylation pattern different from the isolated natural counterpart protein.
71. A recombinant erythropoietm molecule obtainable by a method according to claims 1-17, 56-63 or by use of a human cell according to claims 64-69.
72. A human cell having a sequence encoding at least one El protein of an adenovirus or a functional derivative,
homologue or fragment thereof in its genome, which cell does not produce structural adenoviral proteins and having a gene encoding a recombinant protein.
73. A human cell according to claim 72 which is derived from PER.C6 as deposited under ECACC no. 96022940
74. A human cell according to claim 72 or 73, which further comprises a sequence encoding E2A or a functional derivative or analogue or fragment thereof m its genome.
75. A human cell according to claim 74, wherein said E2A is temperature sensitive.
76. A cell according to anyone of claims 18-32, 72-76, wherein the endogenous DHFR nucleic acid is at least
functionally deleted.
77. Use of a cell according to claim 76, m a method
according to anyone of claims 1-17, 33, 34, 40-54, 56-63 or a use according to anyone of claims 35, 36, 39, 55, 64-69.
78. A viral protein for use m a vaccine obtainable by a methoα according to any one of claims 1-12, 40-63 or by a use according to any one of claims 64-69, said viral protein being free of any non-human mammalian proteinaceous material.

79. Use of an adenoviral EIB protein or a functional
derivative, homologue and/or fragment thereof having anti apoptotic activity for enhancing the production of a
proteinaceous substance m a eukaryotic cell, said use comprising providing said eukaryotic cell with said EIB protein, derivative, homologue and/or fragment thereof.

80. Use according to claim 79, comprising a cell according to claim 18-32, 72-78.
81. Use according to claim 79 or claim 80, in a method according to anyone of claims 1-17, 33, 34, 40-54, 56-63 or a use according to anyone of claims 35, 36, 39, 55, 64-69.