Vectors and host cells comprising a modified SV40 promoter for protein expression

Abstract

The present disclosure is directed to expression vectors, comprising a weakened SV40 promoter, and recombinant mammalian cells capable of producing high levels of a polypeptide of interest, methods of generating and using such recombinant mammalian cells.

What is claimed is: 
     
         1 . A vector comprising a dESV40 promoter operably linked to a polypeptide coding sequence. 
     
     
         2 . The vector of  claim 1 , in which the dESV40 promoter corresponds in sequence to SEQ ID NO: 1. 
     
     
         3 . A vector comprising:
 (i) an expression cassette, where the expression cassette comprises a first polynucleotide sequence encoding a first polypeptide of interest operably linked to a first expression promoter capable of effecting expression of the first polypeptide in a mammalian cell; and   (ii) a polynucleotide sequence encoding a selectable marker operably linked to a dESV40 promoter.   
     
     
         4 . The vector of  claim 3 , in which the expression cassette further comprises a second polynucleotide sequence encoding a second polypeptide of interest operably linked to a second expression promoter capable of effecting expression of the second polypeptide in a mammalian cell. 
     
     
         5 . The vector of  claim 4 , in which the first and second expression promoters are the same. 
     
     
         6 . The vector of  claim 3 ,  4 , or  5 , in which the first and the second expression promoters are selected from the group consisting of: Rous sarcoma virus (RSV) promoter, cytomegalovirus immediate early (CMV IE) promoter, cytomegalovirus major intermediate-early (MIE) promoter, simian virus 40 (SV40) early promoter, Adenovirus major late promoter (AML), mouse mammary tumor virus LTR promoter, Herpes thymidine kinase promoter, human β-actin (ACTB) promoter, elongation factor-1α (EF1α) promoter, the phosphoglycerate kinase (PGK) promoter, the ubiquitinC (UbC) promoter, and murine metallotheionin promoter. 
     
     
         7 . The vector of  claim 3 ,  4 , or  5 , in which the selectable marker is selected from the group consisting of a dihydrofolate reductase (DHFR) and xanthine-guanine phosphoribosyltransferase (XGPRT). 
     
     
         8 . The vector of  claim 3 ,  4 , or  5 , in which the dESV40 promoter corresponds in sequence to SEQ ID NO:1. 
     
     
         9 . The vector of  claim 3 , in which the expression cassette further comprises a second polynucleotide sequence encoding a second polypeptide, 3′ to the first polynucleotide sequence. 
     
     
         10 . The vector of  claim 9 , in which the first promoter is selected from the group consisting of: Rous sarcoma virus (RSV) promoter, cytomegalovirus immediate early (CMV IE) promoter, cytomegalovirus major intermediate-early (MIE) promoter, simian virus 40 (SV40) early promoter, Adenovirus major late promoter (AML), mouse mammary tumor virus LTR promoter, Herpes thymidine kinase promoter, human β-actin (ACTB) promoter, elongation factor-1α (EF1α) promoter, the phosphoglycerate kinase (PGK) promoter, the ubiquitinC (UbC) promoter, and murine metallotheionin promoter. 
     
     
         11 . The vector of  claim 9 , comprising an internal ribosome entry site (IRES) between the first and second polynucleotide sequences. 
     
     
         12 . The vector of  claim 9 ,  10 , or  11 , in which the selectable marker is selected from the group consisting of a dihydrofolate reductase (DHFR) and xanthine-guanine phosphoribosyltransferase (XGPRT). 
     
     
         13 . The vector of  claim 9 ,  10 , or  11 , in which the dESV40 promoter corresponds in sequence to SEQ ID NO:1. 
     
     
         14 . A vector useful for effecting expression of an antibody of interest, comprising:
 (i) an expression cassette, where the expression cassette comprises a first polynucleotide sequence encoding a heavy chain of an antibody of interest, operably linked to a first expression promoter capable of effecting expression of the encoded heavy chain in a mammalian cell, and a second polynucleotide sequence encoding a light chain of an antibody of interest, operably linked to a second expression promoter capable of effecting expression of the encoded light chain in a mammalian cell; and   (ii) a polynucleotide sequence encoding a selectable marker operably linked to a dESV40 promoter.   
     
     
         15 . The vector of  claim 14 , in which the first and second expression promoters are the same and are selected from the group consisting of: Rous sarcoma virus (RSV) promoter, cytomegalovirus immediate early (CMV IE) promoter, cytomegalovirus major intermediate-early (MIE) promoter, simian virus 40 (SV40) early promoter, Adenovirus major late promoter (AML), mouse mammary tumor virus LTR promoter, Herpes thymidine kinase promoter, human β-actin (ACTB) promoter, elongation factor-1α (EF1α) promoter, the phosphoglycerate kinase (PGK) promoter, the ubiquitinC (UbC) promoter, and murine metallotheionin promoter. 
     
     
         16 . The vector of  claim 15 , in which the dESV40 promoter corresponds in sequence to SEQ ID NO:1. 
     
     
         17 . The vector of  claim 14 ,  15 , or  16 , in which the heavy and light chains encoded correspond to antibodies other than elotuzumab. 
     
     
         18 . The vector of  claim 14 ,  15 , or  16 , in which the antibody of interest is other than an antibody having a heavy chain variable region and a light chain variable region of elotuzumab. 
     
     
         19 . The vector of any one of the preceding claims, further comprising an enhancer element operably linked 5′ to the expression promoter. 
     
     
         20 . A mammalian cell that has been transfected with a vector according to any one of  claims 3  to  19 . 
     
     
         21 . A mammalian cell stably transfected with a vector according to any one of  claims 3  to  19 . 
     
     
         22 . The mammalian cell of  claim 20  or  21 , which is a Chinese Hamster Ovary (CHO) cell. 
     
     
         23 . The mammalian cell of  claim 20  or  21 , which is a mouse myeloma NS0 cell. 
     
     
         24 . The mammalian cell of  claim 20  or  21 , which is capable of producing at least 0.5 g/L of the polypeptide of interest in a 10-day fed batch culture. 
     
     
         25 . The mammalian cell of  claim 20  or  21 , which is capable of producing at least 10 pg/cell/day of the polypeptide of interest in a 10-day fed batch culture. 
     
     
         26 . A method of obtaining a mammalian cell capable of producing a polypeptide of interest in high yield in culture, comprising transfecting a mammalian cell with the vector of any one of  claims 3  to  19 , and selecting a mammalian cell that is capable of producing at least 10 pg/cell/day of the polypeptide of interest in a 10-day fed batch culture. 
     
     
         27 . The method of  claim 26 , in which the cell is not subjected to amplification. 
     
     
         28 . A method for producing a polypeptide of interest, comprising culturing the mammalian cell of any one of  claims 20  to  25 . 
     
     
         29 . The method of  claim 28 , wherein the mammalian cell is cultured under conditions that result in the production of at least 10 pg/cell/day of the polypeptide of interest in a 10-day fed batch culture. 
     
     
         30 . The method of  claim 28 , wherein the mammalian cell is cultured under conditions that result in the production of at least 0.5 g/L of the polypeptide of interest in a 10-day fed batch culture.