Efficient production of f(ab')2 fragments in mammalian cells

Abstract

The present invention provides immortalized eukaryotic cells and methods useful for the production of immunologically active bivalent antibody fragments, such as F(ab′)2 fragments. The methods and cells of the invention result in a desirable ratio of bivalent to monovalent antibody fragments.

1 - 24 . (canceled)  
     
     
         25 . An isolated mammalian cell comprising: 
 a nucleic acid sequence encoding an E1 region of adenovirus; and    one or more nucleic acid sequences capable of driving expression operably linked to one or more sequences encoding one or more bivalent multimeric antibody fragments lacking at least the C-terminal constant domain of the heavy chain.    
     
     
         26 . The isolated mammalian cell of  claim 25 , wherein the one or more bivalent multimeric antibody fragments comprises a F(ab′) 2  fragment.  
     
     
         27 . The isolated mammalian cell of  claim 25 , wherein the mammalian cell is of human origin.  
     
     
         28 . The isolated mammalian cell of  claim 25 , wherein the isolated mammalian cell is a retinal cell.  
     
     
         29 . The isolated mammalian cell of  claim 27 , wherein the mammalian cell is a PER.C6™ cell containing the one or more nucleic acid sequences capable of driving expression operably linked to the one or more sequences encoding the one or more bivalent multimeric antibody fragments lacking at least the C-terminal constant domain of the heavy chain.  
     
     
         30 . The isolated mammalian cell oflaim  25 , wherein the one or more sequences capable of driving expression comprise a cytomegalovirus (CMV) promoter.  
     
     
         31 . The isolated mammalian cell of  claim 30 , wherein the CMV promoter comprise a CMV immediate early gene promoter/enhancer.  
     
     
         32 . A mammalian cell comprising an isolated mammalian cell that expresses and secretes both bivalent and monovalent antibody fragments, wherein an elution volume ratio of secreted bivalent antibody fragments to secreted monovalent antibody fragments lacking at least the C-terminal constant domain of the heavy chain is at least 1:3, as may be determined by gel filtration chromatography and analysis of the ratio of an area under the F(ab′) 2  fragment elution peak relative to the F(ab′) elution peak.  
     
     
         33 . The mammalian cell of  claim 32 , wherein the antigen binding regions of the secreted bivalent antibody fragments are not linked by a peptide bond.  
     
     
         34 . The mammalian cell of  claim 32 , wherein the secreted bivalent antibody fragments are immunologically active and is post-translationally modified.  
     
     
         35 . The mammalian cell of  claim 25 , wherein at least one of the one or more nucleic acid sequences is inserted into the plasmid designated pcDNA3002(Neo) as deposited at the ECACCC under accession number 01121318  
     
     
         36 . The proteinaceous molecule of  claim 25 , wherein at least one of the one or more nucleic acid sequences encoding one or more bivalent multimeric antibody fragments lacking at least the C-terminal constant domain of the heavy chain comprises an element selected from the group consisting of at least one of an intron, secreation signal, nuclear localization sequence, proteolytic processing site, glycosylation site, lipid attachment site, sulfation site, site for disulfide bond formation, hydroxylation site, and a phosphorylation site.  
     
     
         37 . A proteinaceous molecule comprising a bivalent antibody fragment, the proteinaceous molecule being produced by a process comprising: 
 providing a mammalian cell comprising: 
 at least one nucleic acid sequence encoding an E1 region of an adenovirus; and  
 at least one nucleic acid encoding at least one bivalent multimeric antibody fragment, wherein the at least one nucleic acid encoding at least one bivalent multimeric antibody fragment lacking at least the C-terminal constant domain of the heavy chain is operably linked to at least one promoter;  
   culturing the mammalian cell;    expressing the at least one nucleic acid encoding at least one bivalent multimeric antibody fragment to produce a proteinaceous molecule; and    purifying the proteinaceous molecule.    
     
     
         38 . The proteinaceous molecule of  claim 37 , wherein culturing the mammalian cell comprises culturing the mammalian cell in a media lacking added serum proteins.  
     
     
         39 . The proteinaceous molecule of  claim 37 , wherein the proteinaceous molecule is secreted from the mammalian cell.  
     
     
         40 . The proteinaceous molecule of  claim 37 , wherein the proteinaceous molecule is immunologically active and is post-translationally modified.  
     
     
         41 . The proteinaceous molecule of  claim 37 , wherein the bivalent antibody fragment's antigen binding regions are not linked by a peptide bond.  
     
     
         42 . The proteinaceous molecule of  claim 37 , wherein the bivalent antibody fragment comprises an F(ab′) 2  fragment.  
     
     
         43 . The proteinaceous molecule of  claim 37 , where the proteinaceous molecule is produced by a method essentially devoid of treating the proteinaceous molecule with a protease.  
     
     
         44 . The proteinaceous molecule of  claim 37 , wherein the mammalian cell is of human origin.  
     
     
         45 . The proteinaceous molecule of  claim 44 , wherein the mammalian cell is of retinal cell origin.  
     
     
         46 . The proteinaceous molecule of  claim 45 , wherein the mammalian cell is a PER.C6™ cell containing the at least one nucleic acid encoding at least one bivalent multimeric antibody fragment lacking at least the C-terminal constant domain of the heavy chain.  
     
     
         47 . The proteinaceous molecule of  claim 37 , wherein the at least promoter comprises a cytomegalovirus (CMV) promoter.  
     
     
         48 . The proteinaceous molecule of  claim 47 , wherein the CMV promoter comprises the CMV immediate early gene enhancer/promoter.  
     
     
         49 . A composition comprising the proteinaceous molecule of  claim 37  together with a pharmaceutically acceptable carrier.  
     
     
         50 . A process for producing a proteinaceous molecule comprising a bivalent antibody fragment, wherein the antigen binding regions of the bivalent antibody fragment are not linked by a peptide bond, the process comprising: 
 providing a mammalian cell comprising: 
 a nucleic acid sequence encoding an E1 region of an adenovirus;  
 one or more nucleic acid sequences capable of driving expression operably linked to one or more nucleotide sequences encoding one or more bivalent multimeric antibody fragments lacking at least the C-terminal constant domain of the heavy chain;  
   culturing the mammalian cell in a suitable medium; and    expressing the one or more nucleic acids to produce the one or more bivalent multimeric antibody fragments.    
     
     
         51 . The process of  claim 50 , further comprising purifying the proteinaceous molecule.  
     
     
         52 . The process of  claim 50 , further comprising secreting the proteinaceous molecule from the mammalian cell.  
     
     
         53 . The process of  claim 50 , wherein the proteinaceous molecule is immunologically active and is post-translationally modified.  
     
     
         54 . The process of  claim 50 , wherein the bivalent antibody fragment comprises a F(ab′) 2  fragment.  
     
     
         55 . The process of  claim 51 , wherein the proteinaceous molecule is produced by a process essentially devoid of treating the proteinaceous molecule with a protease.  
     
     
         56 . The process of  claim 50 , wherein the mammalian cell is of human origin.  
     
     
         57 . The process of  claim 56 , wherein the mammalian cell is a retinal cell.  
     
     
         58 . The process of  claim 57 , wherein the mammalian cell is a PER.C6™ cell containing the one or more nucleic acid sequences capable of driving expression operably linked to the one or more nucleotide sequences encoding the one or more bivalent multimeric antibody fragments.  
     
     
         59 . The process of  claim 50 , wherein the one or more nucleic acid sequences capable of driving expression comprise a nucleic acid sequence from a cytomegalovirus (CMV) promoter.  
     
     
         60 . The process of  claim 59 , wherein the CMV promoter comprises the CMV immediate early gene enhancer/promoter.  
     
     
         61 . The process of  claim 50 , wherein an elution volume ratio of the proteinaceous molecule to monovalent antibody fragments produced by the process is at least 1:3, as may be determined by gel filtration chromatography and analysis of the ratio of an area under the F(ab′) 2  fragment elution peak relative to the F(ab′) elution peak.  
     
     
         62 . The process of  claim 50 , wherein an elution volume ratio of the proteinaceous molecule to monovalent antibody fragments produced by the process is at least 3:1, as may be determined by gel filtration chromatography and analysis of the ratio of an area under the F(ab′) 2  fragment elution peak relative to the F(ab′) elution peak.  
     
     
         63 . The process of  claim 50 , wherein the one or more nucleotide sequences encoding one or more bivalent multimeric antibody fragments comprises an element selected from the group consisting of at least one intron, secretion signal, and nuclear localization sequence.  
     
     
         64 . The process of  claim 50 , wherein the one or more bivalent multimeric antibody fragments comprises an element selected from the group consisting of at least one proteolytic processing site, glycosylation site, lipid attachment site, sulfation site, site for disulfide bond formation, hydroxylation site, and a phosphorylation site  
     
     
         65 . The process of  claim 50 , wherein the one or more bivalent multimeric antibody fragments' antigen binding regions are linked by at least one, but not more than 10 disulfide bonds.  
     
     
         66 . The process of  claim 65 , wherein the antigen binding regions are linked by one or two disulfide bonds.  
     
     
         67 . The process of  claim 65 , wherein the one or more bivalent multimeric antibody fragments' antigen binding regions linked by at least one, but not more than 10 disulfide bonds are not of IgG3 origin.  
     
     
         68 . A method of obtaining an F(ab′) 2  fragment, the method comprising: 
 introducing into a mammalian cell a nucleic acid encoding a F(ab′) 2  fragment, wherein the nucleic acid is operably linked to a sequence that drives expression of the nucleic acid;    culturing the mammalian cell to produce the F(ab′) 2  fragment, wherein an elution volume ratio of the F(ab′) 2  fragment to a F(ab′) produced by the process is at least 1:3, as may be determined by gel filtration chromatography and analysis of the ratio of an area under the F(ab′) 2  fragment elution peak relative to the F(ab′) elution peak; and    isolating the F(ab′) 2  fragment, wherein the isolation is essentially devoid of treating the F(ab′) 2  fragment with a protease.    
     
     
         69 . A vector comprising: 
 a first nucleic acid having a cytomegalovirus (CMV) promoter and a bovine growth hormone polyadenylation signal operably linked to a first sequence encoding a VH1, CH1 and hinge region; and    a second nucleic acid having a CMV immediate early gene enhancer/promoter and a bovine growth hormone polyadenylation signal operably linked to a second sequence encoding a VL and CL region.    
     
     
         70 . The vector of  claim 69 , wherein the first nucleic acid comprises an intron.  
     
     
         71 . The vector of  claim 69 , wherein the second nucleic acid comprises an intron.  
     
     
         72 . A plasmid designated pcDNA3002(Neo) as deposited at the ECACCC under accession number 01121318.