Method for producing monoclonal antibodies

Abstract

An improved method for the production of monoclonal antibodies is disclosed.

1 - 6 . (canceled) 
     
     
         7 . A method of optimizing the expression level of an antibody or a fragment thereof, which comprises:
 (a) producing a eukaryotic host cell containing and capable of expressing a first DNA sequence encoding at least an antibody heavy chain or an antigen binding portion thereof, said first DNA sequence being associated with a first selectable amplifiable marker gene, and a second DNA sequence encoding at least an antibody light chain or an antibody binding portion thereof, said second DNA sequence being associated with said first selectable amplifiable marker gene;   (b) culturing said host cell in a suitable culture medium;   (c) measuring the relative amounts of said first and second DNA sequences expressed;   (d) introducing into said host cell a third DNA sequence encoding at least an antibody chain or antigen binding portion thereof that is limiting, said third DNA sequence being associated with a second selectable amplifiable marker gene; and   (e) differentially amplifying said third DNA sequence to allow production of the desired amount of antibody.   
     
     
         8 . A method of optimizing the expression level of an antibody or a fragment thereof, which comprises:
 (a) producing a eukaryotic host cell containing and capable of expressing a first DNA sequence encoding at least an antibody heavy chain or an antigen binding portion thereof, said first DNA sequence being associated with a first selectable amplifiable marker gene, and a second DNA sequence encoding at least an antibody light chain or an antibody binding portion thereof, said second DNA sequence being associated with said first selectable amplifiable marker gene;   (b) culturing said host cell in a suitable culture medium;   (c) introducing into said host cell a third DNA sequence selected from:
 (i) a DNA sequence encoding at least an antibody heavy chain or antigen binding portion thereof; and 
 (ii) a DNA sequence encoding at least an antibody light chain or antigen binding portion thereof, said third DNA sequence being associated with a second selectable amplifiable marker gene; and 
   (d) differentially amplifying said third DNA sequence to allow production of the desired amount of antibody.   
     
     
         9 . A method of optimizing the expression level of an antibody or a fragment thereof, which comprises:
 (a) producing a eukaryotic host cell containing and capable of expressing a first DNA sequence encoding at least an antibody heavy chain or an antigen binding portion thereof, said first DNA sequence being associated with a first selectable amplifiable marker gene, and a second DNA sequence encoding at least an antibody light chain or an antibody binding portion thereof, said second DNA sequence being associated with said a second selectable amplifiable marker gene;   (b) culturing said host cell in a suitable culture medium;   (c) measuring the relative amounts of said first and second DNA sequences expressed;   (d) introducing into said host cell a third DNA sequence encoding at least an antibody chain or antigen binding fragment thereof that is in limiting amount, said third DNA sequence being associated with a third selectable amplifiable marker gene; and   (e) differentially amplifying said third DNA sequence to allow production of the desired amount of antibody.   
     
     
         10 . The method of  claim 7 , wherein said third DNA sequence encodes an antibody heavy chain or antigen binding portion thereof. 
     
     
         11 . The method of  claim 7 , wherein said third DNA sequence encodes an antibody light chain or antigen binding portion thereof. 
     
     
         12 . The method of  claim 7 , wherein the antibody expression level is about 60 μg/106 cells/48 hrs. 
     
     
         13 . The method of  claim 7 , wherein the first selectable amplifiable marker gene is an ADA gene or a DHFR gene. 
     
     
         14 . The method of  claim 7 , wherein the second selectable amplifiable marker gene is an ADA gene or a DHFR gene. 
     
     
         15 . The method of  claim 9 , wherein the third selectable amplifiable marker gene is an ADA gene or a DHFR gene. 
     
     
         16 . The method of  claim 7 , wherein the first selectable amplifiable marker gene is a DHFR gene and the second selectable amplifiable marker gene is an ADA gene. 
     
     
         17 . The method of  claim 7 , wherein the first selectable amplifiable marker gene is an ADA gene and the second selectable amplifiable marker gene is a DHFR gene. 
     
     
         18 . The method of  claim 7 , wherein the antibody or fragment thereof is a monoclonal antibody. 
     
     
         19 . The method of  claim 7 , wherein the antibody or fragment thereof is a genetically engineered antibody. 
     
     
         20 . The method of  claim 19 , wherein the genetically engineered antibody or fragment thereof is a chimeric, humanized or a CDR-swapped antibody. 
     
     
         21 . The method of  claim 7 , wherein the antibody fragment is selected from the group consisting of Fv, Fab, and F(ab)′2. 
     
     
         22 . The method of  claim 9 , wherein the antibody or fragment thereof is a chimeric protein. 
     
     
         23 . The method of  claim 22 , wherein the chimeric protein comprises a Fab linked to a non-antibody sequence. 
     
     
         24 . The method of  claim 22 , wherein the chimeric protein is a Fab-enzyme or a Fab-toxin. 
     
     
         25 . The method of  claim 9 , wherein the host cell is a mammalian cell. 
     
     
         26 . The method of  claim 9 , wherein the host cell is a non-lymphoid cell. 
     
     
         27 . The method of  claim 26 , wherein the non-lymphoid cell is selected from the group consisting of Chinese Hamster Ovary (CHO) cell, HeLa cells, human 293 cell, COS monkey cell, Bowes cell, mouse L-929 cell, 3T3 cell line, BHK hamster cell, and HaK hamster cell. 
     
     
         28 . The method of  claim 26 , wherein the non-lymphoid cell is a CHO cell. 
     
     
         29 . The method of  claim 9 , wherein the host cell is a lymphocyte-derived cell line. 
     
     
         30 . The method of  claim 29 , wherein the lymphocyte-derived cell line is a murine hybridoma SP2/0-Ag14 or a murine myeloma cell. 
     
     
         31 . The method of  claim 9 , wherein the first DNA sequence is a cDNA or genomic DNA. 
     
     
         32 . The method of  claim 9 , wherein the second DNA sequence is a cDNA or genomic DNA. 
     
     
         33 . The method of  claim 9 , wherein the third DNA sequence is a cDNA or a genomic DNA. 
     
     
         34 . The method of  claim 9 , wherein the first, second, and third DNA sequences are stably integrated into the eukaryotic host cell chromosomal DNA. 
     
     
         35 . The method of  claim 9 , wherein the first and second selectable amplifiable marker gene sequences are stably integrated into the eukaryotic host cell chromosomal DNA. 
     
     
         36 . The method of  claim 9 , wherein the first and second DNA sequences are contained in separate vectors. 
     
     
         37 . The method of  claim 9 , wherein the first and second selectable amplifiable markers are in separate vectors from the first and second DNA sequences. 
     
     
         38 . The method of  claim 9 , wherein the first selectable amplifiable marker is in the same vector as the first DNA sequence. 
     
     
         39 . The method of  claim 9 , wherein the second selectable amplifiable marker is in the same vector as the second DNA sequence. 
     
     
         40 . The method of  claim 36 , wherein the vectors containing the first and second DNA sequences are cotransformed into the eukaryotic host cell. 
     
     
         41 . The method of  claim 9 , wherein said third DNA sequence is introduced into said host cell by fusing said host cell with a host cell comprising said third DNA sequence.