Expression and secretion system

Patent metadata

Field	Value
Publication number	US-9803191-B2
Application number	US-201313934570-A
Country	US
Kind code	B2
Filing date	Jul 3, 2013
Priority date	Jul 5, 2012
Publication date	Oct 31, 2017
Grant date	Oct 31, 2017

Abstract

The invention provides an expression and secretion system, and methods of using the same, for the expression and secretion of one fusion protein in prokaryotic cells and a second fusion protein in eukaryotic cells. Also provided herein are nucleic acid molecules, vectors and host cells comprising such vectors and nucleic acid molecules.

First claim

What is claimed is:

1. An isolated nucleic acid molecule encoding a first polypeptide, a second polypeptide, a signal sequence, and a coat protein or adaptor protein, wherein:
(a) the first polypeptide comprises a variable light chain (VL) domain comprising a VL-HVR1, a VL-HVR2, and a VL-HVR3;
(b) the second polypeptide comprises a variable heavy chain (VH) domain comprising a VH-HVR1, a VH-HVR2, and a VH-VHR3;
(c) the signal sequence is functional in both a prokaryotic cell and an eukaryotic cell and is encoded by a nucleic acid sequence that is 5′ to the nucleic acid sequence encoding the first polypeptide and 5′ to the nucleic acid sequence encoding the second polypeptide, wherein the signal sequence is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11 (consensus mBIP sequence, X ATG AAN TTN ACN GTN GTN GCN GCN GCN CTN CTN CTN CTN GGN GCN GTN CGN GCN, wherein N=A, T, C or G, and wherein X =ATG, X=ATG ACC or X is absent), SEQ ID NO: 16 (Opt1, ATG ATG AAA TTT ACC GTT GTT GCT GCT GCT CTG CTA CTT CTT GGA GCG GTC CGC GCA), SEQ ID NO: 17 (Opt2, ATG ATG AAA TTT ACT GTT GTT GCG GCT GCT CTT CTC CTT CTT GGA GCG GTC CGC GCA), or SEQ ID NO: 18 (Opt 3, ATG ATG AAA TTT ACT GTT GTC GCT GCT GCT CTT CTA CTT CTT GGA GCG GTC CGC GCA); and
(d) the first polypeptide and the second polypeptide form a full-length antibody wherein the first and/or second polypeptide is fused to a coat protein or an adaptor protein.

2. The nucleic acid molecule of claim 1 , wherein the nucleic acid sequence encoding the signal sequence is operably linked to the 5′ end of the nucleic acid sequence encoding the first polypeptide and the 5′ end of the nucleic acid sequence encoding the second polypeptide.

3. The nucleic acid molecule of claim 1 , wherein the VL domain and the VH domain are each linked to a utility peptide.

4. The nucleic acid molecule of claim 3 , wherein the VH domain is linked to a CH1 domain and the VL domain is linked to a CL domain.

5. The nucleic acid molecule of claim 4 , wherein the utility peptide is selected from the group consisting of a Fc, a tag, a label, and a control protein.

6. The nucleic acid molecule of claim 5 , wherein the VL domain is linked to a control protein and the VH domain is linked to the Fc.

7. The nucleic acid molecule of claim 6 , wherein the control protein is a gD protein, or a fragment thereof.

8. The nucleic acid molecule of claim 1 , wherein the nucleic acid encoding the first polypeptide or the second polypeptide is fused to a synthetic intron, wherein the synthetic intron comprises a nucleic acid encoding the coat protein or adaptor protein.

9. The nucleic acid molecule of claim 8 , wherein the synthetic intron is located between a nucleic acid encoding a VH domain and a nucleic acid encoding an Fc or a hinge.

10. The nucleic acid molecule of claim 9 , wherein the synthetic intron further comprises a nucleic acid encoding a naturally occurring intron of IgG1.

11. The nucleic acid molecule of claim 10 , wherein the naturally occurring intron is selected from the group consisting of intron 1, intron 2, and intron 3of IgG1.

12. The nucleic acid molecule of claim 11 , wherein the naturally occurring intron is intron 1 of IgG1.

13. The nucleic acid molecule of claim 8 , wherein the nucleic acid encodes an adaptor protein, and wherein the adaptor protein is a leucine zipper.

14. The nucleic acid molecule of claim 8 , wherein the nucleic acid encodes an adaptor protein, and wherein the adaptor protein comprises the amino acid sequence of SEQ ID NO: 8, 9, 12, 13, 14 or 15.

15. The nucleic acid molecule of claim 8 , wherein the nucleic acid encodes a coat protein, and wherein the coat protein is selected from the group consisting of pI, pII, pIII, pIV, pV, pVI, pVII, pVIII, pIX, and pX of bacteriophage M13, f1, or fd.

16. The nucleic acid molecule of claim 15 , wherein the coat protein is pIII, or a fragment thereof.

17. The nucleic acid molecule of claim 16 , wherein the pIII fragment is amino acids 267-421 or 262-418 of the pIII protein.

18. The nucleic acid molecule of claim 15 , wherein a first fab fusion protein is expressed in prokaryotic cells and a second fab fusion protein is expressed in eukaryotic cells.

19. The nucleic acid molecule of claim 18 , wherein the first fab fusion protein and the second fab fusion protein are the same.

20. The nucleic acid molecule of claim 18 , wherein the first fab fusion protein and the second fab fusion protein are different.

21. The nucleic acid molecule of claim 20 , wherein the first fab fusion protein is a Fab-phage fusion protein.

22. The nucleic acid molecule of claim 21 , wherein the Fab-phage fusion protein comprises VH/CH1 fused to the pIII.

23. The nucleic acid molecule of claim 22 , wherein the second fusion is a Fab-Fc or a Fab-hinge-Fc fusion protein.

24. The nucleic acid molecule of claim 23 , wherein the Fab-Fc or the Fab-hinge-Fc fusion protein comprises VH/CH1 fused to Fc.

25. The nucleic acid molecule of claim 4 , wherein the CH1 domain comprises a portion of a natural splice donor sequence.

26. The nucleic acid molecule of claim 5 , wherein the Fc comprises a portion of a natural splice acceptor sequence.

27. The nucleic acid molecule of claim 4 , wherein the CH1 domain comprises a portion of a splice donor sequence, wherein the portion of the splice donor sequence comprises a modification of at least one nucleic acid residue, and wherein the modification increases splicing efficiency of the nucleic acid molecule relative to a control construct comprising an unmodified splice donor sequence.

28. The nucleic acid molecule of claim 8 , wherein the nucleic acid encodes an adaptor protein, and wherein the adaptor protein comprises an amino acid sequence of SEQ ID NO: 6 (ASIARLRERVKTLRARNYELRSRANMLRERVAQLGGC) or SEQ ID NO: 7 (ASLDELEAEIEQLEEENYALEKEIEDLEKELEKLGGC).

29. A vector comprising the nucleic acid molecule of claim 1 .

30. A cultured host cell transformed with the vector of claim 29 .

31. The cultured host cell of claim 30 wherein the cultured host cell is a bacteria cell.

32. The cultured host cell of claim 31 , wherein the bacteria cell is an E. coli cell.

33. The cultured host cell of claim 30 , wherein the cultured host cell is a eukaryotic cell.

34. The cultured host cell of claim 33 , wherein the eukaryotic cell is a yeast cell, a CHO cell, a 293 cell, or a NSO cell.

35. A process for producing an antibody comprising culturing the cultured host cell of claim 30 , wherein the antibody is expressed.

36. The process of claim 35 , further comprising recovering the antibody expressed by the cultured host cell.

37. The process of claim 36 , wherein the antibody is recovered from the cultured host cell culture medium.

38. The nucleic acid molecule of claim 8 , wherein the synthetic intron is located between a nucleic acid encoding a CH2 domain and a CH3 domain of the antibody.

39. The nucleic acid molecule of claim 8 , wherein the synthetic intron is located between a nucleic acid encoding a hinge region and a CH2 domain of the antibody.

Assignees

• Genentech Inc

Inventors

• Tesar Devin
• Chen Xiaocheng
• Dennis Mark
• Hotzel Isidro

Classifications

• C12N2820/55

  from bacteria · CPC title

• C12N2820/10

  multiple origins of replication · CPC title

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12N2830/55

  from bacteria · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title