Expression vector organization, novel production cell generation methods and their use for the recombinant production of polypeptides

Abstract

Herein is reported an expression vector comprising —an antibody light chain expression cassette, —an antibody heavy chain expression cassette, and —a selection marker expression cassette, wherein the expression cassettes are arranged unidirectional, and wherein the expression cassettes are arranged in the 5′ to 3′ sequence of antibody heavy chain expression cassette, antibody light chain expression cassette and selection marker expression cassette. Further are reported herein methods for the generation of antibody producing cells and the use of these cells for the recombinant production of antibodies.

1 - 52 . (canceled) 
     
     
         53 . An expression vector, comprising:
 an antibody light chain expression cassette;   an antibody heavy chain expression cassette; and   a selection marker expression cassette,   
       wherein the expression cassettes are arranged unidirectional, 
       wherein the expression cassettes are arranged in the 5′ to 3′ sequence of antibody heavy chain expression cassette, antibody light chain expression cassette and selection marker cassette, and 
       wherein one, two or all three expression cassettes comprise the human CMV promoter. 
     
     
         54 . The expression vector according to  claim 53 , wherein the expression cassettes do not comprise a terminator sequence. 
     
     
         55 . The expression vector according to  claim 54 , wherein the terminator sequence is the human gastrin gene transcription terminator sequence (hGT). 
     
     
         56 . The expression vector according to  claim 53 , wherein the antibody light chain expression cassette and/or the antibody heavy chain expression cassette and/or the selection marker expression cassette comprise independently of each other the human CMV promoter. 
     
     
         57 . The expression vector according to  claim 53 , wherein one, two or three expression cassettes comprise the bovine growth hormone polyA signal sequence. 
     
     
         58 . The expression vector according to  claim 53 , wherein the antibody light chain expression cassette and/or the antibody heavy chain expression cassette and/or the selection marker expression cassette comprise independently of each other the bovine growth hormone polyA signal sequence. 
     
     
         59 . The expression vector according to  claim 53 , wherein the antibody light chain expression cassette and/or the antibody heavy chain expression cassette and/or the selection marker expression cassette comprise independently of each other the polyA signal sequence selected from bovine growth hormone polyA signal sequence and the SV40 polyA signal sequence. 
     
     
         60 . The expression vector according to  claim 53 , wherein one, two or all three expression cassettes comprise the human gastrin terminator sequence after the polyA signal sequence. 
     
     
         61 . The expression vector according to  claim 53 , wherein the antibody light chain expression cassette and/or the antibody heavy chain expression cassette and/or the selection marker expression cassette comprise independently of each other the human gastrin terminator sequence after the polyA signal sequence. 
     
     
         62 . The expression vector according to  claim 53 , wherein the antibody light chain expression cassette and/or the antibody heavy chain expression cassette and/or the selection marker expression cassette comprise independently of each other in the 5′ to 3′ direction the bovine growth hormone polyA signal sequence and the human gastrin terminator sequence. 
     
     
         63 . The expression vector according to  claim 53 , wherein the promoter of one, two or all three expression cassettes comprises an Intron A. 
     
     
         64 . The expression vector according to  claim 53 , wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron. 
     
     
         65 . The expression vector according to  claim 53 , wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA. 
     
     
         66 . A method for the selection of a eukaryotic cell comprising a nucleic acid encoding an antibody, wherein a selection agent is added to the cultivation medium for the first time about 24 hours after transfection. 
     
     
         67 . A method for the production of an antibody, comprising the steps of:
 cultivating a eukaryotic cell that has been transfected with an expression vector according to  claim 53 ; and   recovering the antibody from the cell or the cultivation medium.   
     
     
         68 . A method for the production of an antibody, comprising the steps of:
 cultivating a eukaryotic cell that has been transfected with an expression vector encoding an antibody, which has been selected by the addition of a selection agent about 24 hours after transfection to the cultivation; and   recovering the antibody from the cell or the cultivation medium.   
     
     
         69 . The expression vector according to  claim 53 , wherein the expression vector is encoding a bispecific antibody. 
     
     
         70 . The expression vector according to  claim 69 , wherein the bispecific antibody has a first binding specificity or binding site that specifically binds to a first antigen or a first epitope on a first antigen, and the bispecific antibody has a second binding specificity or binding site that specifically binds to a second antigen or second epitope on a second antigen. 
     
     
         71 . The expression vector according to  claim 53 , wherein the expression vector comprises either:
 a first expression cassette comprising in 5′ to 3′ direction a promoter, a nucleic acid encoding a first antibody light chain, a polyA signal sequence, and optionally a terminator sequence,   a second expression cassette comprising in 5′ to 3′ direction a promoter, a nucleic acid encoding a second antibody light chain, a polyA signal sequence, and optionally a terminator sequence,   a third expression cassette comprising in 5′ to 3′ direction a promoter, a nucleic acid encoding a first antibody heavy chain, a polyA signal sequence, and optionally a terminator sequence,   a fourth expression cassette comprising in 5′ to 3′ direction a promoter, a nucleic acid encoding a second antibody heavy chain, a polyA signal sequence, and optionally a terminator sequence,   
       or
 a first expression cassette comprising in 5′ to 3′ direction a promoter, a nucleic acid encoding an antibody light chain, a polyA signal sequence, and optionally a terminator sequence, 
 a second expression cassette comprising in 5′ to 3′ direction a promoter, a nucleic acid encoding a first antibody heavy chain, a polyA signal sequence, and optionally a terminator sequence, and 
 a third expression cassette comprising in 5′ to 3′ direction a promoter, a nucleic acid encoding a second antibody heavy chain, a polyA signal sequence, and optionally a terminator sequence, 
 
       wherein the antibody light chain is a common light chain for both antibody heavy chains. 
     
     
         72 . The expression vector according to  claim 53 , wherein the expression vector comprises:
 an antibody light chain expression cassette,   a first antibody heavy chain expression cassette,   a second antibody heavy chain expression cassette, and   a selection marker expression cassette,   
       wherein at least one of the antibody heavy chain expression cassettes and the antibody light chain expression cassette and the selection marker expression cassette are arranged unidirectional, and 
       wherein the unidirectional expression cassettes are arranged in the 5′ to 3′ sequence of antibody heavy chain expression cassette, antibody light chain expression cassette and selection marker expression cassette. 
     
     
         73 . The expression vector according to  claim 53 , wherein the expression vector comprises:
 a first antibody light chain expression cassette,   a second antibody light chain expression cassette,   a first antibody heavy chain expression cassette,   a second antibody heavy chain expression cassette, and   a selection marker expression cassette,   
       wherein one of the antibody heavy chain expression cassettes and one of the antibody light chain expression cassettes and the selection marker expression cassette are arranged unidirectional, and 
       wherein the unidirectional expression cassettes are arranged in the 5′ to 3′ sequence of antibody heavy chain expression cassette, antibody light chain expression cassette and selection marker expression cassette. 
     
     
         74 . The expression vector according to  claim 53 , wherein one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a hole mutation. 
     
     
         75 . The expression vector according to  claim 53 , wherein one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a knob mutation. 
     
     
         76 . The expression vector according to  claim 53 , wherein one of the antibody light chain expression cassettes encodes an antibody light chain variant comprising an antibody light chain variable domain and an antibody heavy chain CH1 domain as constant domain and/or one of the antibody light chain expression cassettes encodes an antibody light chain comprising an antibody light chain variable domain and an antibody light chain CL domain as constant domain. 
     
     
         77 . The expression vector according to  claim 53 , wherein one of the antibody heavy chain expression cassettes encodes an antibody heavy chain variant comprising as first constant domain an antibody light chain constant domain (CL), and/or one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising as first constant domain an antibody heavy chain CH1 domain.