Recombinant expression of proteins from secretory cell lines

• US 6,194,176 B1
• Filed: 01/17/1997
• Issued: 02/27/2001
• Est. Priority Date: 01/19/1996
• Status: Expired due to Fees

• Current Assignee: Betagene Incorporated, Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Betagene Incorporated, Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Normington, Karl D., Clark, Samuel A., Quaade, Christian, Thigpen, Anice E., Kruse, Fred, Halban, Philippe, Newgard, Christopher B.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/785,271
• Time in Patent Office: 1,502 Days
• Field of Search: 435/69.4, 435/240.2, 435/240.22, 435/252.3
• US Class Current: 435/69.4
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• 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 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/605 Glucagons
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/203 having an IRES
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases

Abstract

The present invention a provides methods for production of heterologous polypeptides using a variety recombinantly engineered secretory cell lines. The common feature of these cell lines is the absence of expression of at least one endogenous polypeptide. The host cell machinery normally used to produce the endogenous polypeptide is then usurped for the purpose of making the heterologous polypeptide. Also described are methods engineering cells for high level expression, methods of large scale protein production, and methods for treatment of disease in vivo using viral delivery systems and recombinant cell lines.

Claims

1. A method for the production of human insulin comprising the steps of:

(a) providing a secretory host cell;

(b) transforming said host cell with an exogenous polynucleotide comprising a gene encoding human insulin, wherein said gene is under the control of a promoter active in eukaryotic cells; and

(c) culturing said secretory host cell under conditions such that said exogenous polynucleotide encoding human insulin expresses human insulin;

wherein said secretory host cell secretes between about 200 ng and about 1000 ng of human insulin/10⁶ cells per hour.

2. The method of claim 1, further comprising blocking the production of an endogenous, secreted polypeptide.

15. The method of claim 1, wherein the insulin is secreted at a rate of about 200 ng of human insulin per 1×

10⁶ cells per hour.

16. The method of claim 1, wherein the insulin is secreted at a rate of about 300 ng of human insulin per 1×

10⁶ cells per hour.

17. The method of claim 1, wherein the insulin is secreted at a rate of about 400 ng of human insulin per 1×

10⁶ cells per hour.

18. The method of claim 1, wherein the insulin is secreted at a rate of about 500 ng of human insulin per 1×

10⁶ cells per hour.

19. The method of claim 1, wherein the insulin is secreted at a rate of about 1000 ng of human insulin per 1×

10⁶ cells per hour.

20. The method of claim 1, wherein said promoter is selected from the group consisting of CMV, SV40 IE, RSV LTR, GAPHD and RIP1.

21. The method of claim 1, wherein said exogenous polynucleotide further comprises an adenovirus tripartite 5′

leader sequence and intron.

22. The method of claim 21, wherein said intron comprises the 5′

donor site of the adenovirus major late transcript and the 3′

splice site of an immunoglobulin gene.

23. The method of claim 1, wherein said exogenous polynucleotide further comprises a polyadenylation signal.

24. The method of claim 1, wherein said secretory host cell is a neuroendocrine cell.

25. The method of claim 24, wherein said insulin is a fusion protein.

30. The method of claim 2, wherein said endogenous, secreted polypeptide is insulin.

31. The method of claim 1, wherein said exogenous polynucleotide further encodes a second exogenous polypeptide that enhances the production and/or secretion of at least one polypeptide produced by said cell.

32. The method of claim 1, wherein said step (b) further comprises contacting said secretory host cell with a polynucleotide comprising a gene for a selectable marker and step (c) further comprises culturing under drug selection.

33. The method of claim 32, wherein said selectable marker gene is flanked by LoxP sites.

34. The method of claim 33, further comprising:

(d) contacting the secretory host cell with a polynucleotide encoding the Cre protein, wherein said polynucleotide is under the control of a promoter active in eukaryotic cells; and

(e) replicate culturing said cell with and without drug selection.

35. The method of claim 32, wherein said selectable marker is hygromycin resistance and said drug is hygromycin.

36. The method of claim 32, wherein said selectable marker is neomycin and said drug is G418.

37. The method of claim 32, wherein said selectable marker is GLUT-2 and said drug is streptozotocin.

38. The method of claim 32, wherein the genes for said insulin polypeptide and said selectable marker are part of the same polynucleotide.

39. The method of claim 1, wherein said secretory host cell is glucose-responsive.

40. The method of claim 2, wherein said blocking of production of said endogenous, secreted polypeptide is effected by interruption of the gene encoding said endogenous, secreted polypeptide.

41. The method of claim 40, wherein said interruption is effected by homologous recombination.

42. The method of claim 24, wherein said secretory host cell is an insulinoma cell.

43. The method of claim 42, wherein said insulinoma cell is a rat insulinoma cell.

44. The method of claim 42, wherein said insulinoma cell is a human insulinoma cell.

45. The method of claim 31, wherein said exogenous polypeptides is selected from the group consisting of a protein processing enzyme, a receptor and a transcription factor.

46. The method of claim 45, wherein said exogenous polypeptide is selected from the group consisting of hexokinase, glucokinase, GLUT-2, GLP-1, IPF1, PC2, PC3, PAM, glucagon-like peptide I receptor, glucose-dependent insulinotropic polypeptide receptor, BIR, SUR, GHRFR and GHRHR.

47. The method of claim 38, wherein the genes for said exogenous insulin and said selectable marker are separated on the same polynucleotide by an internal ribosome entry site.

48. The method of claim 1, wherein said secretory host cell is not glucose-responsive.

49. The method of claim 2, wherein said blocking of production of said endogenous, secreted polypeptide is effected by expression of an RNA antisense to the DNA or mRNA of said endogenous, secreted polypeptide.

50. The method of claim 2, wherein said blocking of production of said endogenous, secreted polypeptide is effected by expression of a ribozyme specific for the mRNA of said endogenous, secreted polypeptide.

51. The method of claim 40, wherein said interruption is effected by genomic site-directed mutagenesis.

52. The method of claim 40, wherein said interruption is effected by random integration.

3. A secretory host cell comprising an exogenous polynucleotide comprising a gene encoding human insulin, wherein said cell secretes between about 200 ng and about 1000 ng of insulin/10⁶ cells per hour.

4. The secretory host cell of claim 3, wherein said exogenous polynucleotide is inserted into the coding or regulatory region of an endogenous, secreted polypeptide, wherein the expression of said endogenous polypeptide is blocked.

5. The secretory host cell of claim 4, wherein said exogenous polynucleotide further comprises a promoter active in eukaryotic cells.

6. The secretory host cell of claim 5, wherein said promoter is selected from the group consisting of CMV, SV40 IE, RSV LTR, GAPHD and RIP1.

7. The secretory host cell of claim 3, wherein said exogenous polynucleotide further comprises an adenovirus tripartite 5′

leader sequence and intron.

8. The secretory host cell of claim 7, wherein said intron comprises the 5′

donor site of the adenovirus major late transcript and the 3′

splice site of an immunoglobulin gene.

9. The secretory host cell of claim 5, wherein said exogenous polynucleotide further comprises a polyadenylation signal.

10. The secretory host cell of claim 3, wherein said cell secretes about 200 ng of human insulin per 1×

10⁶ cells per hour.

11. The secretory host cell of claim 3, wherein said cell secretes about 300 ng of human insulin per 1×

10⁶ cells per hour.

12. The secretory host cell of claim 3, wherein said cell secretes about 400 ng of human insulin per 1×

10⁶ cells per hour.

13. The secretory host cell of claim 3, wherein said cell secretes about 500 ng of human insulin per 1×

10⁶ cells per hour.

14. The secretory host cell of claim 3, wherein said cell secretes about 1000 ng of human insulin per 1×

10⁶ cells per hour.

26. The method of claim 11, wherein said fusion protein comprises a leader sequence that is not naturally associated with said insulin.

27. The method of claim 6, wherein said exogenous polynucleotide further encodes a second exogenous polypeptide wherein said second exogenous polypeptide is amidated, a hormone or a growth factor.

28. The method of claim 27, wherein said amidated polypeptide is selected from the group consisting of calcitonin, calcitonin gene related peptide (CGRP), β

-calcitonin gene related peptide, hypercalcemia of malignancy factor (1-40) (PTH-rP), parathyroid hormone-related protein (107-139) (PTH-rP), parathyroid hormone-related protein (107-111) (PTH-rP), cholecystokinin (27-33) (CCK), galanin message associated peptide, preprogalanin (65-105), gastrin I, gastrin releasing peptide, glucagon-like peptide (GLP-1), pancreastatin, pancreatic peptide, peptide YY, PHM, secretin, vasoactive intestinal peptide (VIP), oxytocin, vasopressin (AVP), vasotocin, enkephalins, enkephalinamide, metorphinamide (adrenorphin), alpha melanocyte stimulating hormone (alpha-MSH), atrial natriuretic factor (5-28) (ANF), amylin, amyloid P component (SAP-1), corticotropin releasing hormone (CRH), growth hormone releasing factor (GHRH), luteinizing hormone-releasing hormone (LHRH), neuropeptide Y, substance K (neurokinin A), substance P and thyrotropin releasing hormone (TRH).

29. The method of claim 27, wherein said hormone is selected from the group consisting of growth hormone, prolactin, placental lactogen, luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-stimulating hormone, leptin, adrenocorticotropin (ACTH), angiotensin I, angiotensin II, β

-endorphin, β

-melanocyte stimulating hormone (β

-MSH), cholecystokinin, endothelin I, galanin, gastric inhibitory peptide (GIP), glucagon, lipotropins, neurophysins and somatostatin.

53. The secretory host cell of claim 5, wherein said exogenous polynucleotide further comprises a gene encoding a second exogenous polypeptide.

54. The secretory host cell of claim 53, wherein the genes encoding said first and said second exogenous polypeptides are separated by an internal ribosome entry site.

55. The secretory host cell of claim 54, wherein said second exogenous polypeptide is a selectable marker.

56. The secretory host cell of claim 55, wherein said selectable marker is hygromycin resistance.

57. The secretory host cell of claim 55, wherein said selectable marker is neomycin.

58. The secretory host cell of claim 55, wherein said selectable marker is GLUT-2.

59. The method of claim 27, wherein said growth factor is selected from the group consisting of epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, hepatocyte growth factor and insulin-like growth factor 1.