Organic compounds

Abstract

The present invention relates to a novel selection system for use in a eukaryotic cell culture process and for expression of a recombinant product of interest. The selection system is based on the introduction of an exogenous functional membrane-bound folate receptor gene together with the polynucleotide or gene encoding the product of interest into a eukaryotic cell and can be widely utilized with eukaryotic cells for which cellular viability is dependent upon folic acid uptake.

1 . A eukaryotic expression vector for recombinant expression of a product of interest in a mammalian cell comprising a first polynucleotide encoding a functional membrane-bound folate receptor serving as selectable marker in a cell culture medium having a limiting concentration of a folate and a second polynucleotide encoding a product of interest to be recombinantly expressed, wherein the product of interest is a pharmaceutically active, therapeutically active, or a diagnostic polypeptide. 
     
     
         2 . The expression vector according to  claim 1 , wherein the first polynucleotide and the second polynucleotide are under the control of distinct transcription promoters. 
     
     
         3 . The expression vector according to  claim 2 , wherein the transcription promoters are the same. 
     
     
         4 . The expression vector according to  claim 2 , wherein the transcription promoters are different. 
     
     
         5 . The expression vector according to  claim 2 , wherein the promoter controlling the transcription of the first polynucleotide is an SV40 promoter. 
     
     
         6 . The expression vector according to  claim 1 , wherein the first polynucleotide and the second polynucleotide are under the control of a common transcription promoter. 
     
     
         7 . The expression vector according to  claim 6 , wherein the common transcription promoter is an SV40 promoter. 
     
     
         8 . The expression vector according to  claim 6 , wherein said vector comprises an IRES element functionally located between the first polynucleotide and the second polynucleotide. 
     
     
         9 . The expression vector according to  claim 1 , wherein the functional membrane-bound folate receptor encoded by the first polynucleotide is selected from the group consisting of a folate receptor alpha (FRα), a folate receptor beta (FRβ), and a functional mutant thereof. 
     
     
         10 . The expression vector according to  claim 9 , wherein the functional membrane-bound folate receptor encoded by the first polynucleotide is a human folate receptor alpha (hFRα). 
     
     
         11 . A mammalian cell for recombinantly producing a product of interest wherein the cellular viability of said cell is dependent on folate uptake, and into which mammalian cell a first polynucleotide located on an expression vector and encoding a functional membrane-bound folate receptor serving as selectable marker in a cell culture medium having a limiting concentration of a folate and a second polynucleotide located on an expression vector and encoding a product of interest have been stably introduced, wherein the first polynucleotide and the second polynucleotide are located on the same expression vector or on separate an expression vectors and wherein the product of interest is a pharmaceutically active, therapeutically active, or diagnostic polypeptide. 
     
     
         12 . The mammalian cell according to  claim 11 , wherein said cell is lacking full activity of at least one endogenous functional membrane-bound folate receptor. 
     
     
         13 . The mammalian cell according to  claim 11 , wherein said first polynucleotide encoding a functional membrane-bound folate receptor and said second polynucleotide encoding a product of interest are located on the same expression vector. 
     
     
         14 . The mammalian cell according to  claim 11 , wherein the expression vector is a eukaryotic expression vector for recombinant expression of a product of interest in a mammalian cell comprising a first polynucleotide encoding a functional membrane-bound folate receptor serving as selectable marker in a cell culture medium having a limiting concentration of a folate and a second polynucleotide encoding a product of interest to be recombinantly expressed, wherein the product of interest is a pharmaceutically or therapeutically active or a diagnostic polypeptide. 
     
     
         15 . The mammalian cell according to  claim 11 , wherein said mammalian cell is a rodent cell. 
     
     
         16 . The mammalian cell according to  claim 15 , wherein said rodent cell is a CHO cell. 
     
     
         17 . A process for production of a mammalian cell according to  claim 13 , said process comprising providing a mammalian cell for which cellular viability is dependent upon folate uptake, and introducing a first polynucleotide located on an expression vector and encoding the functional membrane-bound folate receptor serving as selectable marker in a cell culture medium having a limiting concentration of a folate and a second polynucleotide located on an expression vector and encoding the product of interest, wherein the first polynucleotide and the second polynucleotide are located on the same expression vector or on separate an expression vectors. 
     
     
         18 . The process according to  claim 17 , wherein the first polynucleotide and the second polynucleotide are located on the same expression vector. 
     
     
         19 . The process according to  claim 18 , wherein the expression vector is a eukaryotic expression vector for recombinant expression of a product of interest in a mammalian cell comprising a first polynucleotide encoding a functional membrane-bound folate receptor serving as selectable marker in a cell culture medium having a limiting concentration of a folate and a second polynucleotide encoding a product of interest to be recombinantly expressed, wherein the product of interest is a pharmaceutically or therapeutically active or a diagnostic polypeptides. 
     
     
         20 . A method for selection of a eukaryotic cell capable of stably expressing a product of interest encoded by an expression vector which has been introduced into the cell, comprising
 (i) providing a plurality of eukaryotic cells for which cellular viability is dependent upon folate uptake, and into which cells a first polynucleotide located on an expression vector and encoding a functional membrane-bound folate receptor and a second polynucleotide located on an expression vector and encoding the product of interest have been introduced, wherein the first polynucleotide and the second polynucleotide are located on the same expression vector or on separate expression vectors,   (ii) culturing said plurality of eukaryotic cells in a cell culture medium having a limiting concentration of a folate, thereby obtaining a eukaryotic cell wherein stable expression of the product of interest is achieved.   
     
     
         21 . The method according to  claim 20 , further comprising identifying and isolating a eukaryotic cell wherein stable expression of the product of interest is achieved. 
     
     
         22 . The method according to  claim 20 , wherein the plurality of eukaryotic cells is composed of eukaryotic cells as defined in  claim 11 . 
     
     
         23 . A process for production of a product of interest, comprising
 (i) performing a method of selection according to  claim 11 , and   (ii) isolating the product of interest from said cell culture medium or from said cell.   
     
     
         24 . A method for selection of a eukaryotic cell capable of stably expressing a product of interest comprising the use of a functional membrane bound folate receptor as a section marker, wherein said eukaryotic cell cellular viability is dependent on the uptake of folate. 
     
     
         25 . The method according to  claim 24 , wherein the folate receptor is selected from the group consisting of the folate receptor alpha (FRα), the folate receptor beta (FRβ), and a functional mutant thereof. 
     
     
         26 . The method according to  claim 25 , wherein the folate receptor is the human folate receptor alpha (hFRα).