Modular assembly of antibody genes, antibodies prepared thereby and use

• US 5,618,920 A
• Filed: 04/29/1994
• Issued: 04/08/1997
• Est. Priority Date: 11/01/1985
• Status: Expired due to Term

• Current Assignee: Xoma Technology Ltd. (Xoma Corp.)
• Original Assignee: Xoma Corp.
• Inventors: Horwitz, Arnold H., Robinson, Randy R., Lei, Shau-Ping, Liu, Alvin Y., Wall, Randolph, Better, Marc, Wilcox, Gary L.
• Primary Examiner(s): Budens, Robert D.
• Assistant Examiner(s): BURKE, JULIE ELAINE
• Application Number:
  US08/235,225
• Time in Patent Office: 1,075 Days
• Field of Search: 424/133.1, 435/172.1, 435/172.3, 435/240.1, 435/240.27, 435/252.3, 435/252.33, 530/387.3, 530/387.1
• US Class Current: 530/387.1
• 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 the secretion of heavy chain immunoglobulin fragments and light chain immunoglobulins from prokaryotic hosts using a prokaryotic secretion signal peptide wherein the heavy chain fragments and light chains are capable of associating to form an antigen binding antibody fragment.

Claims

1. A secretable immunoglobulin heavy chain fragment comprising an entire variable region directly linked to a prokaryotic secretion signal peptide, wherein said secretable heavy chain fragment is capable of being secreted from a prokaryotic organism and of providing an immunoglobulin heavy chain fragment that is capable of associating with an immunoglobulin light chain, or with an immunoglobulin light chain fragment that comprises an entire variable region, and, when so associated, of binding antigen.

2. The secretable immunoglobulin heavy chain fragment of claim 1, wherein said fragment is the Fd portion of said heavy chain fragment.

6. The secretable immunoglobulin light chain of any one of claims 1-5, wherein said organism is E. coli.

7. The secretable immunoglobulin light chain of any of claims 1-5, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

8. The secretable immunoglobulin light chain of claim 7, wherein said organism is E. coli.

3. A secretable immunoglobulin light chain, or secretable light chain fragment comprising an entire variable region, directly linked to a prokaryotic secretion signal peptide, wherein said secretable light chain or secretable light chain fragment is capable of being secreted from a prokaryotic organism and of providing an immunoglobulin light chain or light chain fragment that is capable of associating with an immunoglobulin heavy chain fragment that comprises an entire variable region, and, when so associated, of binding antigen.

4. The secretable immunoglobulin light chain of claim 3, wherein said immunoglobulin chain is a full-length light chain.

5. The secretable immunoglobulin light chain of claim 3, wherein said immunoglobulin chain is said light chain fragment.

9. A method of producing an immunoglobulin heavy chain fragment comprising an entire variable region, said method comprising the steps of:

1) expressing, in a prokaryotic organism, a polynucleotide sequence encoding a secretable immunoglobulin heavy chain fragment that comprises an entire variable region directly linked to a prokaryotic secretion signal peptide,

2) secreting said secretable immunoglobulin heavy chain fragment from said organism so as to provide an immunoglobulin heavy chain fragment that is capable of associating with an immunoglobulin light chain, or with an immunoglobulin light chain fragment that comprises an entire variable region, and, when so associated, of binding antigen.

10. The method of claim 9, wherein said fragment is the Fd portion of said heavy chain fragment.

11. The method of any one of claims 9-10, wherein said fragment is recovered from the periplasmic space of said organism.

12. The method of claim 11, wherein said organism is E. coli.

13. The method of claim 11, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

14. The method of claim 13, wherein said organism is E. coli.

15. The method of claim 11, wherein said polynucleotide is part of a dicistronic transcription unit that further provides polynucleotide sequences encoding a secretable immunoglobulin light chain or light chain fragment, said secretable immunoglobulin light chain or light chain fragment comprising an entire variable region directly linked to a prokaryotic secretion signal peptide.

16. The method of claim 15, wherein said organism is E. coli.

17. The method of claim 15, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

18. The method of claim 17, wherein said organism is E. coli.

19. The method of any one of claims 9-10, wherein said fragment is recovered from the culture medium of said host.

20. The method of claim 19, wherein said organism is E. coli.

21. The method of claim 19, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

22. The method of claim 19, wherein said organism is E. coli.

23. The method of claim 19, wherein said polynucleotide is part of a dicistronic transcription unit that further provides polynucleotide sequences encoding a secretable immunoglobulin light chain or light chain fragment said secretable immunoglobulin light chain or light chain fragment comprising an entire variable region directly linked to a prokaryotic secretion signal peptide.

24. The method of claim 23, wherein said organism is E. coli.

25. The method of claim 23, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

26. The method of claim 25, wherein said organism is E. coli.

27. A method of producing an immunoglobulin light chain or light chain fragment comprising an entire variable region, said method comprising the steps of:

1) expressing, in a prokaryotic organism, a polynucleotide sequence encoding a secretable immunoglobulin light chain, or light chain fragment that comprises an entire variable region, directly linked to a prokaryotic secretion signal peptide,

2) secreting said secretable immunoglobulin light chain or light chain fragment from said organism so as to provide an immunoglobulin light chain, or light chain fragment, that is capable of associating with an immunoglobulin heavy chain fragment that comprises an entire variable region, and, when so associated, of binding antigen.

28. The method of claim 27, wherein said immunoglobulin chain is said light chain.

29. The method of claim 27, wherein said immunoglobulin chain is said light chain fragment.

30. The method of any one of claims 27-29, wherein said immunoglobulin chain or said fragment is recovered from the periplasmic space of said organism.

31. The method of claim 30, wherein said organism is E. coli.

32. The method of claim 30, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

33. The method of claim 32, wherein said organism is E. coli.

34. The method of claim 32, wherein said polynucleotide is part of a dicistronic transcription unit that further provides polynucleotide sequence encoding a secretable immunoglobulin heavy chain fragment or immunoglobulin Fd, said secretable immunoglobulin heavy chain fragment or Fd comprising an entire variable region directly linked to a prokaryotic secretion signal peptide.

35. The method of claim 34, wherein said organism is E coli.

36. The method of claim 34, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

37. The method of claim 36, wherein said organism is E. coli.

38. The method of any one of claims 27-29, wherein said immunoglobulin chain or said fragment is recovered from the culture medium of said host.

39. The method of claim 38, wherein said organism is E. coli.

40. The method of claim 38, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

41. The method of claim 40, wherein said organism is E. coli.

42. The method of claim 38, wherein said polynucleotide is part of a dicistronic transcription unit that further provides polynucleotide sequences encoding a secretable immunoglobulin heavy chain fragment or immunoglobulin Fd, said secretable immunoglobulin heavy chain fragment or Fd comprising an entire variable region directly linked to a prokaryotic secretion signal peptide.

43. The method of claim 42, wherein said organism is E. coli.

44. The method of claim 42, wherein said prokaryotic secretion signal peptide is a pectate lyase signal peptide.

45. The method of claim 44, wherein said organism is E. coli.