Selection of cells expressing heteromeric polypeptides

Abstract

This invention is in the general field of recombinant expression of polypeptides in animal cell culture. More particularly, the invention concerns improved selection in cells of recombinantly engineered vectors designed to express polypeptides.

1 . A method of selecting cells comprising 
 transfecting cells with a vector comprised of a first nucleic acid encoding a first desired polypeptide, wherein the transcription of said first nucleic acid is operably linked to a second nucleic acid encoding a first subunit of a selectable marker, and further comprising a third nucleic acid encoding a second desired polypeptide that is capable of associating with the first desired polypeptide to form a heteromeric complex, wherein the transcription of said third nucleic acid is operably linked to a fourth nucleic acid which encodes a second subunit of a selectable marker, and wherein said second subunit is capable of associating with the first subunit of the selectable marker, thereby providing a selectable activity    applying selection conditions to the transfected cells and    selecting for cells expressing the heteromeric complex.    
     
     
         2 . The method of  claim 1 , wherein the heteromeric complex is an antibody.  
     
     
         3 . The method of  claim 1 , wherein the first nucleic acid encodes a polypeptide selected from the group consisting of an antibody heavy chain and an antibody light chain.  
     
     
         4 . The method of  claim 1 , wherein the selectable marker is selected from the group consisting of a drug resistance marker, a metabolic survival marker, a color marker and a fluorescent marker.  
     
     
         5 . The method of  claim 4 , wherein the selectable marker is selected from the group consisting of dihydrofolate reductase, neomycin resistance, hygromycin resistance, beta-galactosidase, and green fluorescent protein.  
     
     
         6 . The method of  claim 1 , wherein an internal ribosomal entry site occurs between the first nucleic acid and the second nucleic acid.  
     
     
         7 . The method of  claim 1 , wherein an internal ribosomal entry site occurs between the third nucleic acid and the fourth nucleic acid.  
     
     
         8 . The method of  claim 1 , wherein the selectable marker subunit is a fusion polypeptide comprising an interaction domain.  
     
     
         9 . The method of  claim 8 , wherein the interaction domain is a dimerization sequence that is a leucine zipper from a polypeptide selected from the group consisting of GCN4, C/EBP, c-Fos, c-Jun, c-Myc and c-Max.  
     
     
         10 . The method of  claim 1 , further encoding a different functional selectable marker selected from the list consisting of zeomycin, neomycin, puromycin, Blasticidin S, and GPT.  
     
     
         11 . The host cell of  claim 1 , which is selected from the group consisting of CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, a myeloma cell line, and W138 cells.  
     
     
         12 . A method of selecting cells comprising 
 transfecting an expression system comprising a first vector comprising a first nucleic acid encoding a light chain of an antibody wherein the transcription of said light chain is operably linked to the transcription of a second nucleic that encodes a fusion polypeptide of a first subunit of dihydrofolate reductase fused to a dimerization sequence, and a second vector comprising a third nucleic acid encoding a heavy chain of an antibody wherein the transcription of said heavy chain is operably linked to the transcription of a fourth nucleic acid that encodes a fusion polypeptide of a second subunit of a dihydrofolate reductase fused to a dimerization sequence wherein each subunit of dihydrofolate reductase does not have selectable activity when expressed alone and co-expression of the first dihydrofolate reductase subunit with the second dihydrofolate reductase subunit provides dihydrofolate reductase activity    applying selection conditions to the cells, and    selecting for cells expressing the heteromeric complex.    
     
     
         13 . The expression system of  claim 12 , wherein one subunit of dihydrofolate reductase is amino acids 1 to 105 of SEQ ID NO:5 and the other subunit of dihydrofolate reductase is amino acids 106 to 187 of SEQ ID NO:5.  
     
     
         14 . The expression system of  claim 13 , wherein the dimerization sequence fused to the dihydrofolate reductase subunit is derived from the GCN4 leucine zipper sequence.  
     
     
         15 . A host cell transfected with the expression system of any of claims  12 ,  13 , or  14 .  
     
     
         16 . An isolated nucleic acid molecule comprising a first nucleic acid encoding a polypeptide, wherein said first nucleic acid is operably linked to a second nucleic acid encoding a subunit of a selectable marker, and wherein said subunit or subunits is capable of interacting with a different subunit of the selectable marker thereby providing a selectable activity.