Production of human mutated proteins in human cells by means of homologous recombination

Abstract

The invention concerns a process for the production of muteins of eukaryotic polypeptides in eukaryotic cells by means of homologous recombination. The invention additionally concerns a process for the production of human cells which are suitable for the production of human mutated proteins. Finally the invention concerns the human cells produced by the process and mutated human proteins obtainable therefrom as well as pharmaceutical preparations which contain these muteins.

1 . Process for the production of muteins of eukaryotic polypeptides wherein 
 (i) a nucleic acid molecule capable of homologous recombination is introduced into eukaryotic cells which contain a target nucleic acid sequence coding for an endogenous target polypeptide, the said nucleic acid molecule comprising 
 (a) at least one sequence section which is homologous to sequences in the gene locus of the target nucleic acid sequence and, compared to the endogenous target nucleic acid sequence, has a mutation in the coding region of the mature target polypeptide and  
 (b) a nucleic acid section coding for a selection marker,  
   (ii) the cells are cultured under such conditions that a homologous recombination of the introduced nucleic acid molecule takes place whereby the cell contains a mutated target nucleic acid sequence after the homologous recombination which is able to express a mutein of the target polypeptide,    (iii) the cells, in which a homologous recombination has taken place, are selected and    (iv) the mutein is isolated from the cells or/and the cell supernatant.    
     
     
         2 . Process as claimed in  claim 1 , wherein 
 the cell is a human cell.    
     
     
         3 . Process as claimed in  claim 2 , wherein 
 the cell is a HeLa cell, a Namalwa cell or a HT1080 cell.    
     
     
         4 . Process as claimed in one of the  claims 1  to  3 , wherein 
 a starting cell is used which contains the target nucleic acid sequence on multiple chromosomes.  
 
     
     
         5 . Process as claimed in one of the previous claims, wherein 
 additionally the expression of the target nucleic acid sequence is activated by introducing a heterologous expression control sequence.    
     
     
         6 . Process as claimed in  claim 5 , wherein 
 the heterologous expression control sequence is a viral promoter and in particular a CMV promoter.    
     
     
         7 . Process as claimed in one of the previous claims, wherein 
 the nucleic acid section coding for the selection marker is a neomycin phosphotransferase gene.    
     
     
         8 . Process as claimed in one of the previous claims, wherein 
 the nucleic acid molecule introduced into the cell additionally contains an amplification gene and the mutated target nucleic acid sequence is amplified after the homologous recombination.    
     
     
         9 . Process as claimed in  claim 8 , wherein 
 a dihydrofolate reductase gene is used as an amplification gene.    
     
     
         10 . Process as claimed in one of the previous claims, wherein 
 the target nucleic acid sequence is a tissue plasminogen activator (t-PA), erythropoietin, insulin, tumour necrosis factor, interleukin or interleukin receptor sequence.    
     
     
         11 . Process as claimed in one of the  claims 1  to  9 , wherein 
 the mutein is a polypeptide derived from t-PA comprising the K2 and P domains of t-PA.  
 
     
     
         12 . Process as claimed in one of the previous claims, wherein 
 the mutein is isolated from the supernatant of cells cultured in suspension.    
     
     
         13 . Process as claimed in one of the previous claims, wherein 
 the mutein is isolated from the supernatant of cells cultured in serum-free medium.    
     
     
         14 . Mutated human polypeptide from a human cell obtainable by a process as claimed in one of the  claims 1  to  13 , characterized by human glycosylation and the absence of polypeptides that are foreign to the species.  
     
     
         15 . Process for the production of a human cell which expresses a mutein of a human target polypeptide, wherein 
 (i) a nucleic acid molecule is introduced into human cells which contain a target nucleic acid sequence coding for an endogenous target polypeptide, the said nucleic acid molecule comprising 
 (a) at least one sequence section which is homologous to sequences in the gene locus of the target nucleic acid sequence and, compared to the endogenous target nucleic acid sequence, has a mutation in the coding region of the mature target polypeptide,  
 (b) optionally a heterologous expression control sequence for the target nucleic acid sequence and  
 (c) a nucleic acid section coding for a selection marker,  
   (ii) the cells are cultured under such conditions that a homologous recombination of the introduced nucleic acid molecule takes place whereby the cell contains a mutated target nucleic acid sequence after the homologous recombination which is able to express a mutein of the target polypeptide,    (iii) the cells, in which a homologous recombination has taken place, are selected and    (iv) the cells selected in this way are isolated.    
     
     
         16 . Process as claimed in  claim 15 , wherein 
 the nucleic acid molecule additionally contains an amplification gene and, after the homologous recombination, the mutated target nucleic acid sequence is amplified.    
     
     
         17 . Process as claimed in  claim 16 , wherein 
 a dihydrofolate reductase gene is used as an amplification gene.    
     
     
         18 . Human cell obtainable by a process as claimed in one of the  claims 15  to  17  which contains at least one endogenous gene which codes for a mutated human polypeptide.  
     
     
         19 . Use of a human cell as claimed in  claim 18  for the production of a mutein of a human polypeptide.  
     
     
         20 . Pharmaceutical preparation, wherein 
 it contains a mutein as claimed in  claim 14  as the active substance optionally together with other active substances or/and common pharmaceutical carriers, auxiliary substances or additives.