Separate-cistron contructs for secretion of aglycosylated antibodies from prokaryotes

• US 6,979,556 B2
• Filed: 12/13/2001
• Issued: 12/27/2005
• Est. Priority Date: 12/14/2000
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Simmons, Laura C., Yansura, Daniel G., Reilly, Dorothea, Klimowski, Laura
• Primary Examiner(s): LEFFERS JR, GERALD G
• Application Number:
  US10/020,786
• Publication Number:
  US 20030073164A1
• Time in Patent Office: 1,475 Days
• Field of Search: 435/69.6, 435/69.7, 435/69.1, 435/252.3, 435/252.33, 435/471, 536/23.1, 536/23.4, 530/387.1
• US Class Current: 435/69.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2845 against integrin beta2-subunit-containing molecules, e.g. CD11, CD18
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/36 against blood coagulation factors
• CPC Class Codes: C07K 16/4291 against IgE
• CPC Class Codes: C07K 2317/20 characterized by taxonomic origin
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

The present invention provides methods and compositions for improved expression and production of recombinant antibodies in prokaryotic expression systems. Particularly contemplated are prokaryotic expression and production of full length aglycosylated antibodies. The antibody products of the invention can be used in various aspects of biological research, diagnosis and medical treatment.

Claims

1. A polynucleotide molecule encoding an intact antibody, said polynucleotide molecule comprising (1) a first promoter and a first cistron forming a first promoter-cistron pair and (2) a second promoter and a second cistron forming a second promoter-cistron pair, wherein the first cistron of said first promoter-cistron pair comprises a first translational initiation region (TIR-L) operably linked to a nucleic acid sequence encoding an immunoglobulin light chain and the second cistron of said second promoter-cistron pair comprises a second translational initiation region (TIR-H) operably linked to a nucleic acid sequence encoding an immunoglobulin heavy chain, wherein upon expression of said polynucleotide in a prokaryotic host cell, secreted light and heavy chains are folded and assembled to form a biologically active intact antibody.

2. The polynucleotide molecule of claim 1, wherein the first and second promoters are prokaryotic promoters selected from the group consisting of phoA, tac, lpp, lac-lpp, lac, ara, trp, trc and T7 promoters.

3. The polynucleotide molecule of claim 2, wherein both promoters are PhoA promoters.

4. The polynucleotide molecule of claim 1, wherein each of the TIR-L and TIR-H comprises a prokaryotic secretion signal sequence or variant thereof.

5. The polynucleotide molecule of claim 4, wherein the prokaryotic secretion signal sequence is selected from the group consisting of STII, OmpA, PhoE, LamB, MBP and PhoA secretion signal sequences.

6. The polynucleotide molecule of claim 1, wherein the TIR-L and TIR-H provide approximately equal translational strengths.

7. The polynucleotide molecule of claim 6, wherein the relative translational strength combination is about (1-TIR-L, 1-TIR-H).

8. A recombinant vector for expressing an intact antibody in a prokaryotic host cell, said vector comprising the polynucleotide molecule of claim 1.

9. A prokaryotic host cell comprising the recombinant vector of claim 8.

10. The prokaryotic host cell of claim 9 which is a gram-negative bacterial cell.

11. The host cell of claim 10 which is E. coli.

12. The host cell of claim 11, further comprising a polynucleotide encoding at least one prokaryotic polypeptide selected from the group consisting of DsbA, DsbC, DsbG and FkpA.

13. The host cell of claim 12, wherein the polynucleotide encodes both DsbA and DsbC.

14. The host cell of claim 11, wherein the E. coli is of a strain deficient in endogenous protease activities.

15. The host cell of claim 14, wherein the genotype of the E. coli strain lacks degP and prc genes and harbors a mutant spr gene.

16. A process for producing a biologically active intact antibody in a prokaryotic host cell, said process comprising expressing in the host cell a polynucleotide comprising (1) a first promoter and a first cistron forming a first promoter-cistron pair and (2) a second promoter and a second cistron forming a second promoter-cistron pair, wherein the first cistron of said first promoter-cistron pair comprises a first translational initiation region (TIR-L) operably linked to a nucleic acid sequence encoding an immunoglobulin light chain and the second cistron of said second promoter-cistron pair comprises a second translational initiation region (TIR-H) operably linked to a nucleic acid sequence encoding an immunoglobulin heavy chain, wherein upon expression of said polynucleotide, secreted light chain and heavy chain are folded and assembled to form a biologically active intact antibody;

and recovering said intact antibody.

17. The process of claim 16, wherein the first and the second promoters are prokaryotic promoters selected from the group consisting of phoA, tac, lpp, lac-lpp, lac, ara, trp, trc and T7 promoters.

18. The process of claim 17, wherein both the first and the second promoters are PhoA promoters.

19. The process of claim 16, wherein each of the TIR-L and TIR-H comprises a prokaryotic secretion signal sequence or variant thereof.

20. The process of claim 19, wherein the prokaryotic secretion signal sequence is selected from the group consisting of STII, OmpA, PhoE, LamB, MBP and PhoA secretion signal sequences.

21. The process of claim 16, wherein the TIR-L and TIR-H provide approximately equal translational strengths.

22. The process of claim 21, wherein the relative translational strength combination is about (1-TIR-L, 1-TIR-H).

23. The process of claim 16, wherein the prokaryotic host cell is E. coli.

24. The process of claim 16, further comprising expressing in the prokaryotic host cell a polynucleotide encoding at least one prokaryotic polypeptide selected from the group consisting of DsbA, DsbC, DsbG and FkpA.

25. The process of claim 24, wherein the polynucleotide encodes both DsbA and DsbC.

26. The process of claim 23, wherein the E. coli is of a strain deficient in endogenous protease activities.

27. The process of claim 26, wherein the genotype of the E. coli lacks degP and prc genes and harbors a mutant spr gene.