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 a cytomegalovirus (CMV) promoter comprising nucleotides 1 to 608 of SEQ ID NO: 1; and,
optionally,
(e) a nucleic acid sequence encoding an antibody heavy or light chain constant region, operably linked to the insertion site.
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 3 , 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 chain 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 , wherein the OriP origin of replication comprises nucleotides 1795 to 3545 of SEQ ID NO: 1.
18. The expression vector of claim 1 , wherein the SV40 origin of replication comprises nucleotides 5834 to 6140 of SEQ ID NO: 1.
19. The expression vector of claim 1 , wherein the expression vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 27, 29, and 30.
20. The expression vector of claim 1 , wherein the expression vector is described in a Figure selected from the group consisting of FIGS. 1 , 8 , 10 , and 11 .
21. The expression vector of claim 1 , further comprising a nucleic acid sequence encoding a signal peptide.
22. A kit comprising the vector of claim 1 .
23. A mammalian host cell comprising the vector of claim 1 .
24. The mammalian host cell of claim 23 , wherein the cell is a COS cell or a human embryonic kidney (HEK) cell.
25. The mammalian host cell of claim 24 , wherein the cell is a COST cell.
26. The mammalian host cell of claim 24 , wherein the cell is an HEK-293-6E cell.
27. A method for producing a recombinant protein comprising introducing 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.
28. 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 cytomegalovirus (CMV) promoter operably linked to the insertion site,
wherein the vector comprises the nucleic acid sequence of SEQ ID NO: 1.
29. A kit comprising the vector of claim 28 .
30. A mammalian host cell comprising the vector of claim 28 .
31. A method for producing a recombinant protein comprising introducing the expression vector of claim 28 into a mammalian host cell, culturing the mammalian host cell under suitable conditions so as to express the protein, and recovering the protein.