Modular assembly of antibody genes, antibodies prepared thereby and use

• US 6,204,023 B1
• Filed: 06/06/1995
• Issued: 03/20/2001
• Est. Priority Date: 11/01/1985
• Status: Expired due to Term

• Current Assignee: Xoma Technology Ltd. (Xoma Corp.)
• Original Assignee: Xoma Ltd.
• Inventors: Robinson, Randy R., Liu, Alvin Y., Wall, Randolph, Better, Marc, Wilcox, Gary L., Horwitz, Arnold H., Lei, Shau-Ping
• Primary Examiner(s): BURKE, JULIE ELAINE
• Application Number:
  US08/472,691
• Time in Patent Office: 2,114 Days
• Field of Search: 435/172.3, 435/69.6, 435/69.7, 435/240.27, 435/252.33, 435/71.3, 435/328, 435/91.1, 424/133.1, 424/134.1, 530/387.3, 536/23.53
• US Class Current: 435/71.3
• 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: C07K 14/43 Sweetening agents, e.g. thaumatin, monellin
• CPC Class Codes: C07K 16/3046 Stomach, Intestines
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/734 Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/034 containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
• CPC Class Codes: C07K 2319/036 targeting to the medium outside of the cell, e.g. type III secretion
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/88 Lyases (4.)

Abstract

The invention relates to cDNA genetic sequences, vehicles containing same as well as hosts tansformed therewith, for the production of chimeric immunoglobulin molecules, functional fragments thereof and immunoglobulin derivatives exhibiting novel functional properties comprising human constant region modules and non-human variable region modules, or for class switching antibody molecules and/or chains.

The invention also relates to DNA coding for pectate lyase signal peptide has been cloned on a plasmid to create a secretion vector which is capable of producing a chosen protein which is transported across the bacterial membrane. The secretion vector has been used to secrete extracellular thaumatin and extracellular chimeric antibody fragments. The proteins produced by this vector have biological activity. The thaumatin is properly folded and the antibody fragments are capable of binding antigens on target cancer cells. The invention also relates to the secretion of chimeric antibodies and fragments thereof from yeast in functional form.

Claims

1. A process for preparing an immunoglobulin that is capable of binding antigen, said immunoglobulin comprising (a) a heavy chain or heavy chain fragment and (b) a light chain or light chain fragment, said process comprising expressing, in a baceterial cell, of a first nucleic acid that encodes a bacterial signal sequence operably linked to either said heavy chain or to said heavy chain fragment, and a second nucleic acid that encodes a bacterial signal sequence operably linked to either said light chain or to said light chain fragment, such that the bacterial signal sequences bring about the transport of said heavy chain or said heavy chain fragment and said light chain or said light chain fragment through the cytoplasmic membrane of a bacterial cell, and, obtaining said immunoglobulin from the periplasmic space or culture medium, wherein said immunoglobulin is capable of binding antigen.

2. The process of claim 1, wherein (a) is said heavy chain.

3. The process of claim 1, wherein (a) is said heavy chain fragment.

4. The process of claim 3, wherein said heavy chain frament is Fd.

5. The process of claim 1, wherein (b) is said light chain.

6. The process of claim 1, wherein (b) is said light chain fragment.

7. The process of claim 1, wherein (a) is said heavy chain and (b) is said light chain.

8. The process of claim 1, wherein (a) is said heavy chain fragment and (b) is said light chain fragment.

9. The process of claim 1, wherein (a) is said heavy chain fragment and (b) is said light chain.

10. The process of claim 9, wherein said immunoglobulin is Fab.

11. The process of claim 1, wherein one or more chains selected from the group consisting of said heavy chain, said heavy chain fragment, said light chain and said light chain fragment, contains a variable region and a constant region that are from different antibodies.

12. The process of claim 11, wherein said light chain or said light chain fragment contains a variable region and a constant region that are from different antibodies.

13. The process of claim 11, wherein said heavy chain or said heavy chain fragment contains a variable region and a constant region that are from different antibodies.

14. The process of claim 11, wherein each chain in said immunoglobulin contains a variable region and a constant region that are from different antibodies.

15. the process of any one claims 11-14, wherein said variable region is from the same species as that of the constant region.

16. The process of claim 15, wherein said species is human.

17. The process of any one of claims 11-14, wherein said variable region is from a different species than that of the constant region.

18. The process of claim 17, wherein said variable region is from a non-human species.

19. The process of claim 18, wherein said constant region is a human constant region.

20. The process of claim 1 or 2-11, wherein said first nucleic acid and said second nucleic acid comprise a dicistronic transcription unit.

21. The process of any one of claims 1 or 2-11, wherein said first nucleic acid and said second nucleic acid are on the same or different plasmids.

22. The process of claim 21, wherein both said first nucleic acid and said second nucleic acid are on the same plasmid.

23. The process of claim 21, wherein said first nucleic acid is on a different plasmid than said second nucleic acid.

24. The process of any one of claims 1 or 2-11, wherein said immunoglobulin fragment is obtained from said periplasmic space of said bacterial cell.

25. The process of claim 24, wherein said bacterial cell is E. coli.

26. The process of any one of claims 1 or 2-11, wherein said immunoglobulin fragment is obtained from said culture medium.

27. The process of claim 26, wherein said bacterial cell is E. coli.

28. The process of any one of claims 1 or 2-11, wherein said bacterial signal sequence encoded by said first nucleic acid or said bacterial signal sequence encoded by said second nucleic acid, or both, is a pectate lyase signal sequence.

29. The process of claim 28, wherein said bacterial cell is E. coli.

30. The process of any one of claims 1 or 2-11, wherein said nucleic acid encoding said heavy chain or heavy chain fragment, and said light chain or light chain fragment is on a bacteriophage vector.

31. A process for preparing an immunoglobulin that is capable of binding antigen, said immunoglobulin comprising (a) a heavy chain variable region and (b) a light chain variable region, said process comprising operably linking a nucleic acid encoding a bacterial signal sequence to a nucleic acid encoding said heavy chain variable region or said light chain variable region or both of said variable regions such that the bacterial signal sequence or sequences bring about the transport of said variable regions through the cytoplasmic membrane of a bacterial cell, expressing said nucleic acid in said bacterial cell, and obtaining said immunoglobulin from the periplasmic space or culture medium, wherein said immunoglobulin is capable of binding antigen.

32. The process of claim 31, wherein said immunoglobulin comprises a full length heavy chain.

33. The process of claim 31, wherein said immunoglobulin comprises a heavy chain constant region.

34. The process of claim 31, wherein said immunoglobulin comprises Fd.

35. The process of claim 31, wherein said immunoglobulin comprises a full length light chain.

36. The process of claim 31, wherein said immunoglobulin comprises a light chain constant region.

37. The process of claim 31, wherein said immunoglobulin comprises a full length heavy chain and a full length light chain.

38. The process of claim 33, wherein said immunoglobulin comprises a heavy chain constant region and a light chain constant region.

39. The process of claim 31, wherein said immunoglobulin comprises a heavy chain constant region and a full length light chain.

40. The process of claim 39, wherein said immunoglobulin is Fab.

41. The process of claim 31, wherein said immunoglobulin comprises a heavy chain constant region that is from a different antibody than that of said heavy chain variable region or comprises a light chain constant region that is from a different antibody than that of said light chain variable region.

42. The process of claim 41, wherein said light chain constant region is from a different antibody than that of said light chain variable region.

43. The process of claim 41, wherein said heavy chain constant region is from a different antibody than that of said heavy chain variable region.

44. The process of claim 31, wherein said immunoglobulin comprises a heavy chain constant region that is from a different antibody than said heavy chain variable region and a light chain constant region that is from a different antibody than that of said light chain variable region.

45. The process of claim 41 or 44, wherein said light chain variable region is from the same species as that of said light chain constant region or said heavy chain variable region is from the same species as that of said heavy chain constant region.

46. The process of claim 45, wherein said species is human.

47. The process of claim 41 or 44, wherein said light chain variable region is from a different species than that of said light chain constant region or said heavy chain variable region is from a different species than that of said heavy chain region.

48. The process of claim 47, wherein said light chain variable region or said heavy chain variable region is from a non-human species.

49. The process of claim 48, wherein said light chain constant region or said heavy chain constant region is a human constant region.

50. The process of any one of claims 31-41, wherein said nucleic acid encoding said light chain variable region and said heavy chain variable region comprises a dicistronic transcription unit.

51. The process of any one of claims 31-41, wherein said nucleic acid encoding said light chain variable region and said heavy chain variable region are on the same or different plasmids.

52. The process of claim 51, wherein said nucleic acid encoding said light chain variable region and said heavy chain variable region are on the same plasmid.

53. The process of claim 51, wherein said nucleic acid encoding said light chain variable region and said heavy chain variable region are on different plasmids.

54. The process of any one claims 31-41, wherein said immunoglobulin is obtained from said periplasmic space of said bacterial cell.

55. The process of claim 54, wherein said bacterial cell is E. coli.

56. The process of any one claims 31-41, wherein said immunoglobulin is obtained from said culture medium.

57. The process of claim 56, wherein said bacterial cell is E. coli.

58. The process of any one of claims 31-41, wherein said bacterial signal sequence that is operably linked to said light chain variable region or said heavy chain variable region, or both, is a pectate lyase signal sequence.

59. The process of claim 58, wherein said bacterial cell is E. coli.

60. The process of any one of claims 31-41, wherein said nucleic acid encoding said variable regions is on a bacteriophage vector.