Methods for treating patients with adenoviral vectors

• US 20030008375A1
• Filed: 12/27/2001
• Published: 01/09/2003
• Est. Priority Date: 11/20/1996
• Status: Abandoned Application

• Current Assignee: Crucell Holland B.V (Johnson & Johnson)
• Original Assignee: Introgen Therapeutics, Inc.
• Inventors: Zhang, Shuyuan, Wu, Zheng, Thwin, Capucine, Cho, Toohyon
• Application Number:
  US10/033,491
• Publication Number:
  US 20030008375A1
• Time in Patent Office: Days
• US Class Current: 435/235.1
• CPC Class Codes: A61K 35/761 Adenovirus
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/4746 p53
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10045 Special targeting system for viral vectors
• CPC Class Codes: C12N 2710/10051 Methods of production or purification of viral material
• CPC Class Codes: C12N 2710/10322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2710/10332 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/10351 Methods of production or purification of viral material
• CPC Class Codes: C12N 2810/6018 Adenoviridae
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The present invention addresses the need to improve the yields of viral vectors when grown in cell culture systems. In particular, it has been demonstrated that for adenovirus, the use of low-medium perfusion rates in an attached cell culture system provides for improved yields. In other embodiments, the inventors have shown that there is improved Ad-p53 production witrh cells grown in serum-free conditions, and in particular in serum-free suspension culture. Also important to the increase of yields is the use of detergent lysis. Combination of these aspects of the invention permits purification of virus by a single chromatography step that results in purified virus of the same quality as preparations from double CsCl banding using an ultracentrifuge.

Claims

1. A method for producing an adenovirus comprising:

a) growing host cells in media at a low perfusion rate;

b) infecting said host cells with an adenovirus;

c) harvesting and lysing said host cells to produce a crude cell lysate;

d) concentrating said crude cell lysate;

e) exchanging buffer of crude cell lysate; and

f) reducing the concentration of contaminating nucleic acids in said crude cell lysate.

2. The method of claim 1, further comprising isolating an adenoviral particle from said cell lysate using chromatography.

3. The method of claim 1, wherein the glucose concentration in said media is maintained between about 0.7 and about 1.7 g/L.

4. The method of claim 1, wherein said exchanging buffer involves a diafiltration step.

5. The method of claim 1, wherein said adenovirus comprises an adenoviral vector encoding an exogenous gene construct.

6. The method of claim 5, wherein said gene construct is operatively linked to a promoter.

7. The method of claim 6, wherein said promoter is SV40 IE, RSV LTR, β

-actin, CMV IE, adenovirus major late, polyoma F9-1, or tyrosinase.

8. The method of claim 1, wherein said adenovirus is a replication-incompetent adenovirus.

9. The method of claim 8, wherein the adenovirus is lacking at least a portion of the E1-region.

10. The method of claim 9, wherein the adenovirus is lacking at least a portion of the EIA and/or E1B region.

11. The method of claim 1, wherein said host cells are capable of complementing replication.

12. The method of claim 1, wherein said host cells are 293 cells.

13. The method of claim 5, wherein said exogenous gene construct encodes a therapeutic gene.

14. The method of claim 13, wherein said therapeutic gene encodes antisense ras, antisense myc, antisense raf antisense erb, antisense src, antisensefins, antisense jun, antisense trk antisense ret, antisense gsp, antisense hst, antisense bcl antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF, thymidine kinase or p53.

15. The method of claim 14, wherein said therapeutic gene encodes p53.

16. The method of claim 1, wherein said cells are harvested and lysed ex situ using a hypotonic solution, hypertonic solution, freeze-thaw, sonication, impinging jet, microfluidization or a detergent.

17. The method of claim 1, wherein said cells are harvested and lysed in situ using a hypotonic solution, hypertonic solution, or a detergent.

18. The method of claim 17, wherein said cells are lysed and harvested using detergent.

19. The method of claim 18, wherein said detergent is Thesit®

, NP-40®

, Tween-20®

, Brij-58®

, Triton X®

-100 or octyl glucoside.

20. The method of claim 1, wherein said lysis is achieved through autolysis of infected cells.

21. The method of claim 1, wherein said cell lysate is treated with Benzonase®

, or Pulmozyme®

.

22. The method of claim 2, wherein said isolating consists essentially of a single chromatography step.

23. The method of claim 22, wherein said chromatography step is ion exchange chromatography.

24. The method of claim 23, wherein said ion exchange chromatography is anion exchange chromatography.

25. The method of claim 24, wherein said anion exchange chromatography utilizes DEAE, TMAE, QAE, or PEI.

26. The method of claim 24, wherein said anion exchange chromatography utilizes Toyopearl Super Q 650M, MonoQ, Source Q or Fractogel TMAE.

27. The method of claim 24. wherein said ion exchange chromatography is carried out at a pH range of between about 7.0 and about 10.0.

28. The method of claim 1, further comprising a concentration step employing membrane filtration.

29. The method of claim, 28, wherein said filtration is tangential flow filtration.

30. The method of claim, 28, wherein said filtration utilizes a 100 to 300K NMWC, regenerated cellulose, or polyether sulfone membrane.

31. An adenovirus produced according to a process comprising the steps of:

a) growing host cells in media at a low perfusion rate;

b) infecting said host cells with an adenovirus;

c) harvesting and lysing said host cells to produce a crude cell lysate;

d) concentrating said crude cell lysate;

e) exchanging buffer of crude cell lysate; and

f) reducing the concentration of contaminating nucleic acids in said crude cell lysate.

32. The adenovirus of claim 31, wherein adenovirus comprises an adenoviral vector encoding an exogenous gene construct.

33. The adenovirus of claim 31, wherein said gene construct is operatively linked to a promoter.

34. The adenovirus of claim 31, wherein said adenovirus is a replication-incompetent adenovirus.

35. The adenovirus of claim 34, wherein said adenovirus is lacking at least a portion of the E1-region.

36. The adenovirus of claim 31, wherein the adenovirus is lacking at least a portion of the E1A and/or E1B region.

37. The adenovirus of claim 31, wherein said host cells are capable of complementing replication.

38. The adenovirus of claim 31, wherein said host cells are 293 cells.

39. The adenovirus of claim 31, wherein said exogenous gene construct encodes a therapeutic gene.

40. The adenovirus of claim 39, wherein said therapeutic gene encodes antisense ras, antisense myc, antisense raf antisense erb, antisense src, antisense fis, antisense jun, antisense trk antisense ret, antisense gsp, antisense hst, antisense bcl antisense abl, Rb, CFTR, p16, p21, p27, p57, p73, C-CAM, APC, CTS-1, zac1, scFV ras, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, BRCA1, VHL, MMAC1, FCC, MCC, BRCA2, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF G-CSF, thyrnidine kinase or p53.

41. The adenovirus of claim 40, wherein said therapeutic gene is p53.

42. The adenovirus of claim 33, wherein said promoter is SV40 IE, RSV LTR, β

-actin or CMV IE, adenovirus major late, polyoma F9-1, or tyrosinase.

43. A method for the purification of an adenovirus comprising:

a) growing host cells;

b) infecting said host cells with an adenovirus;

c) harvesting and lysing said host cells by contacting said cells with a detergent to produce a crude cell lysate;

d) concentrating said crude cell lysate;

e) exchanging buffer of crude cell lysate; and

f) reducing the concentration of contaminating nucleic acids in said crude cell lysate.

44. The method of claim 43, further comprising isolating an adenoviral particle from said lysate using chromatography.

45. The method of claim 43, wherein said host cells are grown in media wherein a glucose concentration is maintained between about 0.7 and about 1.7 g/L.

46. The method of claim 43, wherein said exchanging buffer involves a diafiltration step.

47. The method of claim 43, wherein said detergent is Thesit®

, NP-40®

, Tween-20®

, Brij-58®

, Triton X-100 or octyl glucoside.

48. The method of claim 47, wherein said detergent is present in the lysis solution at a concentration of about 1% (w/v).

49. The method of claim 43, wherein said isolating consists essentially of a single chromatography step.

50. The method of claim 44, wherein said chromatography step is ion exchange chromatography.

51. An adenovirus produced according to a process comprising the steps of:

a) growing host cells;

b) infecting said host cells with an adenovirus;

c) harvesting and lysing said host cells by contacting said cells with a detergent to produce a crude cell lysate;

d) concentrating said crude cell lysate;

e) exchanging buffer of crude cell lysate; and

f) reducing the concentration of contaminating nucleic acids in said crude cell lysate.

52. A method for the purification of an adenovirus comprising:

a) growing host cells in serum-free media;

b) infecting said host cells with an adenovirus;

c) harvesting and lysing said host cells to produce a crude cell lysate;

d) concentrating said crude cell lysate;

e) exchanging buffer of crude cell lysate; and

f) reducing the concentration of contaminating nucleic acids in said crude cell lysate.

53. The method of claim 52, wherein said host cells are adapted for growth in serum-free media.

54. The method of claim 52, wherein said cells are grown as a cell suspension culture.

55. The method of claim 52, wherein said cells are grown as an anchorage-dependent culture.

56. The method of claim 53, wherein said adaptation for growth in serum-free media comprises a sequential decrease in the fetal bovine serum content of the growth media.

57. The method of claim 53, wherein said serum-free media comprises a fetal bovine serum content of less than 0.03% v/v.

58. The method of claim 52, further comprising isolating an adenoviral particle from said lysate using chromatography.

59. The method of claim 52, wherein said lysis is achieved through autolysis of infected cells.

60. The method of claim 52, wherein said exchanging buffer involves a diafiltration step.

61. The method of claim 52, wherein said detergent is Thesit®

, NP-40®

, Tween-20®

, Brij-58®

, Triton X-100®

or octyl glucoside.

62. The method of claim 52, wherein said detergent is present in the lysis solution at a concentration of about 1% (w/v).

63. The method of claim 52, wherein said isolating consists essentially of a single chromatography step.

64. The method of claim 58, wherein said chromatography step is ion exchange chromatography.

65. An adenovirus produced according to a process comprising the steps of:

a) growing host cells in serum-free media;

b) infecting said host cells with an adenovirus;

c) harvesting and lysing said host cells to produce a crude cell lysate;

d) concentrating said crude cell lysate;

e) exchanging buffer of crude cell lysate; and

f) reducing the concentration of contaminating nucleic acids in said crude cell lysate.

66. A 293 host cell adapted for growth in serum-free media.

67. The cell of claim 66, wherein said cell is adapted for growth in suspension culture.

68. The cell of claim 66, wherein the cell is deposited with the ATCC and is designated as a IT293SF cell.

69. The cell of claim 66, wherein said adaptation for growth in serum-free media comprises a sequential decrease in the fetal bovine serum content of the growth media.”