Mammalian expression vectors and uses thereof
View Patent ↗The present invention features nucleic acids for recombinant protein expression in mammalian cell culture. The episomal vectors of the invention promote high protein production in mammalian cells expressing the SV40 T Ag or Epstein-Barr virus nuclear antigen (e.g., COS7 or HEK293-6E cells). The methods and systems are useful, for example, in pharmaceutical drug development and cloning, especially for the production of antibodies.
1. An expression vector comprising:
(a) an OriP origin of replication derived from Epstein-Barr virus (EBV);
(b) an SV40 origin of replication;
(c) an insertion site for inserting a gene of interest;
(d) a promoter operably linked to the insertion site, wherein the promoter is an EF-1α promoter comprising nucleotides 76 to 1267 of SEQ ID NO: 2; and, optionally,
(e) a nucleic acid sequence encoding an antibody heavy or light chain constant region, operably linked to the insertion site,
wherein the OriP origin of replication is bound by a trans-acting EBNA1 replication initiation factor that is not encoded by the expression vector.
2. The expression vector of claim 1 , wherein the gene of interest is an antibody heavy or light chain variable region.
3. The expression vector of claim 2 , wherein the antibody heavy or light chain variable region is selected from the group consisting of murine, humanized, chimeric and human.
4. The expression vector of claim 2 , wherein the antibody heavy chain variable region is the heavy chain variable region of an antibody selected from the group consisting of an anti-TNFα antibody, an anti-IL-18 antibody, and an anti-IL-12 antibody.
5. The expression vector of claim 3 , wherein the antibody light chain variable region is the light chain variable region of an antibody selected from the group consisting of an anti-TNFα antibody, an anti-IL-18 antibody, and an anti-IL-12 antibody.
6. The expression vector of claim 1 , wherein the antibody heavy chain constant region is murine or human.
7. The expression vector of claim 1 , wherein the antibody heavy constant region is selected from the group consisting of gamma 1, z, a; gamma 1, z, non-a; gamma 2, n+; gamma 2, n−; and gamma 4.
8. The expression vector of claim 7 , wherein the gamma 1, z, non-a antibody heavy chain constant region further comprises an alanine mutation at position 234 of the heavy chain constant region.
9. The expression vector of claim 8 , further comprising an alanine mutation at either position 235 or 237 of the antibody heavy chain constant region.
10. The expression vector of claim 1 , wherein the antibody light chain constant region is either a human kappa isotype or a human lambda isotype.
11. The expression vector of claim 1 , wherein the antibody heavy chain constant region is either a murine gamma 1 isotype or a murine gamma 2a isotype.
12. The expression vector of claim 1 , wherein the antibody light chain constant region is a murine kappa isotype.
13. The expression vector of claim 1 , wherein the antibody heavy chain constant region is an Fc domain.
14. The expression vector of claim 2 , wherein the heavy or light chain antibody variable region is 5′ to the insertion site.
15. The expression vector of claim 1 , further comprising a selectable marker.
16. The expression vector of claim 15 , wherein the selectable marker is an ampicillin resistance gene.
17. The expression vector of claim 1 , the expression vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 31 and 32.
18. The expression vector of claim 1 , the expression vector comprising the nucleic acid sequence of SEQ ID NO:2.
19. The expression vector of claim 1 , further comprising a nucleic acid sequence encoding a signal peptide.
20. A kit comprising the vector of claim 1 .
21. A mammalian host cell comprising the vector of claim 1 .
22. The mammalian host cell of claim 21 , that is a COS cell or a human embryonic kidney (HEK) cell.
23. The mammalian host cell of claim 22 , which is a COS7 cell.
24. The mammalian host cell of claim 22 , which is an HEK-293-6E cell.
25. A method of producing a recombinant protein comprising introducing the the expression vector of claim 1 into a mammalian host cell, culturing the mammalian host cell under suitable conditions so as to express the protein, and recovering the protein.
26. An expression vector comprising:
(a) an OriP origin of replication derived from Epstein-Barr virus (EBV);
(b) an SV40 origin of replication;
(c) an insertion site for inserting a gene of interest;
(d) an EF-1α promoter operably linked to the insertion site, wherein the EF-1 α promoter comprises nucleotides 76 to 1267 of SEQ ID NO: 2; and, optionally,
(e) a nucleic acid sequence encoding an antibody heavy or light chain constant region, operably linked to the insertion site.
27. The expression vector of claim 26 , wherein the gene of interest is an antibody heavy or light chain variable region.
28. The expression vector of claim 27 , wherein the antibody heavy or light chain variable region is selected from the group consisting of murine, humanized, chimeric and human.
29. The expression vector of claim 27 , wherein the antibody heavy chain variable region is the heavy chain variable region of an antibody selected from the group consisting of an anti-TNFα antibody, an anti-IL-18 antibody, and an anti-IL-12 antibody.
30. The expression vector of claim 27 , wherein the antibody light chain variable region is the light chain variable region of an antibody selected from the group consisting of an anti-TNFα antibody, an anti-IL-18 antibody, and an anti-IL-12 antibody.
31. The expression vector of claim 26 , further comprising the nucleic acid sequence encoding the antibody heavy or light chain constant region, operably linked to the insertion site.
32. The expression vector of claim 31 , wherein the antibody heavy chain constant region is murine or human.
33. The expression vector of claim 31 , wherein the antibody heavy constant region is selected from the group consisting of gamma 1, z, a; gamma 1, z, non-a; gamma 2, n+; gamma 2, n−; and gamma 4.
34. The expression vector of claim 33 , wherein the gamma 1, z, non-a antibody heavy chain constant region further comprises an alanine mutation at position 234 of the heavy chain constant region.
35. The expression vector of claim 34 , further comprising an alanine mutation at either position 235 or 237 of the antibody heavy chain constant region.
36. The expression vector of claim 31 , wherein the antibody light chain constant region is either a human kappa isotype or a human lambda isotype.
37. The expression vector of claim 31 , wherein the antibody heavy chain constant region is either a murine gamma 1 isotype or a murine gamma 2a isotype.
38. The expression vector of claim 31 , wherein the antibody light chain constant region is a murine kappa isotype.
39. The expression vector of claim 31 , wherein the antibody heavy chain constant region is an Fc domain.
40. The expression vector of claim 31 , wherein the heavy or light chain antibody variable region is 5′ to the insertion site.
41. The expression vector of claim 26 , further comprising a selectable marker.
42. The expression vector of claim 41 , wherein the selectable marker is an ampicillin resistance gene.
43. The expression vector of claim 26 , wherein the EF-1α promoter is human.
44. The expression vector of claim 26 , the vector comprising the nucleic acid sequence of SEQ ID NO: 2.
45. A kit comprising the vector of claim 26 .
46. A mammalian host cell comprising the vector of claim 26 .
47. A method of producing a recombinant protein comprising introducing the expression vector of claim 26 into a mammalian host cell, culturing the mammalian host cell under suitable conditions so as to express the protein, and recovering the protein.
48. An expression vector comprising:
(a) an OriP origin of replication derived from Epstein-Barr virus (EBV);
(b) an SV40 origin of replication;
(c) an insertion site for inserting a gene of interest; and
(d) an EF-1α promoter operably linked to the insertion site, wherein the vector comprises the nucleic acid sequence of SEQ ID NO: 2.
49. A kit comprising the vector of claim 48 .
50. A mammalian host cell comprising the vector of claim 48 .
51. A method of producing a recombinant protein comprising introducing the expression vector of claim 48 into a mammalian host cell, culturing the mammalian host cell under suitable conditions so as to express the protein, and recovering the protein.