Methods and Constructs for Expressing Polypeptide Multimers in Eukaryotic Cells Using Alternative Splicing

Abstract

The invention provides a method of producing multiple polypeptides, such as antibodies or antibody fragments, in a eukaryotic cell using a single expression vector. The expression vector is engineered to comprise two or more expression cassettes under the control of a single promoter wherein the expression cassettes have splice sites which allow for their alternative splicing and expression as two or more independent gene products at a desired ratio. Use of the vector for the efficient expression of recombinant antibodies in eukaryotic host cells is disclosed as well as the use of such antibodies in diagnostic and therapeutic applications.

1 - 24 . (canceled) 
     
     
         25 . An expression vector comprising,
 a promoter;   a 5′ UTR;   a single splice donor;   an intron;   a first splice acceptor;   a first exon encoding a first antibody polypeptide or fragment thereof;   a second splice acceptor; and   a second exon encoding a second antibody polypeptide or fragment thereof,   wherein the promoter is operably linked to the first and second exon,   wherein upon entry into a cell, said single splice donor splices with said first splice acceptor, forming a spliced transcript which permits translation of said first exon, and said second splice acceptor, forming a spliced transcript which permits translation of said second exon,   wherein said first antibody polypeptide or fragment thereof and said second antibody polypeptide or fragment thereof are expressed from said spliced transcripts and associate to form an antibody or antibody fragment; and   wherein cells comprising said expression vector produce 10 picograms or more of antibody per cell per day.   
     
     
         26 - 28 . (canceled) 
     
     
         29 . The vector of  claim 25 , wherein the first or second antibody polypeptide, or fragment thereof, or both is murine, chimeric, humanized, human or synthetic. 
     
     
         30 - 34 . (canceled) 
     
     
         35 . The vector of  claim 25 , wherein said vector is a viral vector. 
     
     
         36 . (canceled) 
     
     
         37 . An isolated eukaryotic cell containing the vector of  claim 25 . 
     
     
         38 . The isolated cell of  claim 37 , wherein said vector is integrated into the chromosomal DNA of said cell. 
     
     
         39 . The isolated cell of  claim 37 , wherein said vector is episomal. 
     
     
         40 . The isolated cell of  claim 37 , wherein said cell is a mammalian cell or a yeast cell. 
     
     
         41 . The isolated cell of  claim 40 , wherein said cell is selected from the group comprising: a baby hamster kidney cell, a fibroblast, a myeloma cell, an NS0 cell, a PER.C6 cell, or a Chinese Hamster Ovary (CHO) cell. 
     
     
         42 . The isolated cell of  claim 41 , wherein said cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         43 . A method of producing antibodies or antibody fragments, the method comprising: (a) culturing a cell of  claim 37  in a culture; and (b) isolating said first antibody polypeptide or fragment thereof and said second antibody polypeptide or fragment thereof from the culture. 
     
     
         44 - 46 . (canceled) 
     
     
         47 . The expression vector of  claim 25 , wherein the promoter is a CMV promoter. 
     
     
         48 . An expression vector comprising,
 a promoter;   a 5′ UTR;   a single splice donor;   an intron;   a first splice acceptor;   a first exon encoding a first antibody polypeptide or fragment thereof;   a second splice acceptor; and   a second exon encoding a second antibody polypeptide or fragment thereof,   wherein the promoter is operably linked to the first and second exon,   wherein upon entry into a cell, said single splice donor splices with said first splice acceptor, forming a spliced transcript which permits translation of said first exon, and said second splice acceptor, forming a spliced transcript which permits translation of said second exon,   wherein said first antibody polypeptide or fragment thereof and said second antibody polypeptide or fragment thereof are expressed from said spliced transcripts and associate to form an antibody or antibody fragment; and   wherein cells comprising said expression vector secrete antibody into the cell culture media at a concentration in a range selected from the group consisting of:   (i) 7.6 to 271.3 ng/mL;   (ii) 22 to 229.7 ng/mL;   (iii) 6.9 to 185.5 ng/mL;   (iv) 130 to 151 ng/mL;   (v) 0.5 to 78.8 ng/mL; and,   (vi) 23 to 58 ng/mL.   
     
     
         49 . The vector of  claim 48 , wherein the first or second antibody polypeptide, or fragment thereof, or both is murine, chimeric, humanized, human or synthetic. 
     
     
         50 . The vector of  claim 48 , wherein said vector is a viral vector. 
     
     
         51 . An isolated eukaryotic cell containing the vector of  claim 48 . 
     
     
         52 . The isolated cell of  claim 51 , wherein said vector is integrated into the chromosomal DNA of said cell. 
     
     
         53 . The isolated cell of  claim 51 , wherein said vector is episomal. 
     
     
         54 . The isolated cell of  claim 51 , wherein said cell is a mammalian cell or a yeast cell. 
     
     
         55 . The isolated cell of  claim 54 , wherein said cell is selected from the group comprising: a baby hamster kidney cell, a fibroblast, a myeloma cell, an NS0 cell, a PER.C6 cell, or a Chinese Hamster Ovary (CHO) cell. 
     
     
         56 . The isolated cell of  claim 55 , wherein said cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         57 . A method of producing antibodies or antibody fragments, the method comprising: (a) culturing a cell of  claim 51  in a culture; and (b) isolating said first antibody polypeptide or fragment thereof and said second antibody polypeptide or fragment thereof from the culture.  58 . The expression vector of  claim 48 , wherein the promoter is a CMV promoter.