Mammalian expression vectors and uses thereof

Abstract

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 or a CMV promoter; and, optionally,   (e) a nucleic acid sequence encoding an antibody heavy or light chain constant region that is 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 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 2 , 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 , wherein the expression vector further comprises an alanine mutation at either position 235 or 237 of the 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 variable region is 5′ to the insertion site. 
     
     
         15 . (canceled) 
     
     
         16 . The expression vector of  claim 1 , wherein the expression vector further comprises a selectable marker. 
     
     
         17 . The expression vector of  claim 16 , wherein the selectable marker is an ampicillin resistance gene. 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The expression vector of  claim 1 , wherein the CMV promoter comprises nucleotides 1 to 608 of SEQ ID NO: 1. 
     
     
         22 . The expression vector of  claim 1 , wherein the EF-1α promoter is human. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . The expression vector of  claim 1 , wherein the EF-1α promoter comprises nucleotides 76 to 1267 of SEQ ID NO: 2. 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . The expression vector of  claim 1 , wherein the OriP origin of replication comprises nucleotides 1795 to 3545 of SEQ ID NO: 1. 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . The expression vector of  claim 1 , wherein the SV40 origin of replication comprises nucleotides 5834 to 6140 of SEQ ID NO: 1. 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . An expression vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 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, 27, 28, 29, 30, 31, and 32. 
     
     
         38 . (canceled) 
     
     
         39 . The expression vector of  claim 1 , wherein the expression vector further comprises a nucleic acid sequence encoding a signal peptide. 
     
     
         40 . A kit comprising the expression vector of  claim 1 . 
     
     
         41 . A host cell comprising the expression vector of  claim 1 . 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . A method for producing a recombinant protein comprising introducing the expression vector of  claim 1  into a host cell, culturing the host cell under suitable conditions so as to express the protein, and recovering the protein. 
     
     
         46 . A kit comprising the expression vector of  claim 37 . 
     
     
         47 . A host cell comprising the expression vector of  claim 37 . 
     
     
         48 . A method for producing a recombinant protein comprising introducing the expression vector of  claim 37  into a host cell, culturing the host cell under suitable conditions so as to express the protein, and recovering the protein.