Vertebrate cells and methods for recombinantly expressing a polypeptide of interest

Abstract

The present application pertains inter alia to an isolated vertebrate cell suitable for recombinant expression of a polypeptide of interest, wherein the vertebrate cell is altered to impair the function of the endogenous protease matriptase and wherein said cell comprises at least one heterologous polynucleotide encoding a polypeptide of interest and wherein the polypeptide of interest is secreted by the cell. It was found that using respective vertebrate cells for producing a recombinant polypeptide of interest significantly reduces clipping of the polypeptide of interest that is secreted into the cell culture medium. Also provided are improved production and screening methods.

1 - 23 . (canceled) 
     
     
         24 . An isolated recombinant vertebrate cell suitable for recombinant expression of a polypeptide of interest, wherein the vertebrate cell is altered to impair the effect of matriptase, wherein the effect of matriptase is impaired because functional expression of the matriptase gene is reduced or eliminated in said cell by gene knock-out, gene mutation, gene deletion, gene silencing or a combination thereof, wherein the vertebrate cell comprises at least one heterologous polynucleotide encoding a polypeptide of interest operatively linked to a secretory leader sequence and the polypeptide of interest is secreted from the vertebrate cell, and wherein impairing the effect of matriptase in said cell reduces clipping of the secreted polypeptide of interest. 
     
     
         25 . The isolated vertebrate cell of  claim 24 , wherein the genome of the vertebrate cell is altered to impair the function of endogenous protease matriptase, and/or wherein at least one or all copies of the matriptase gene are deleted or functionally inactivated. 
     
     
         26 . The isolated vertebrate cell of  claim 25 , wherein
 a) the vertebrate cell comprises one or more mutations in at least one copy or all copies of the matriptase gene to provide a non- or less-functional expression product; or   b) the vertebrate cell comprises one or more mutations in the promoter, in the 5′UTR, the 3′UTR and/or other regulatory elements of the matriptase gene.   
     
     
         27 . The isolated vertebrate cell of  claim 26 , wherein said one or more mutations are comprised in a coding region of the matriptase gene and result in a non- or less functional expression product, wherein optionally the one or more mutations are comprised in a polynucleotide sequence of exon 2 of the matriptase gene or wherein the one or more mutations are comprised in a polynucleotide sequence of the matriptase gene that encodes at least part of the catalytic domain of matriptase whereby a non- or less functional expression product is obtained. 
     
     
         28 . The isolated vertebrate cell of  claim 24 , wherein the vertebrate cell is provided as cell clone or cell line. 
     
     
         29 . The isolated vertebrate cell of  claim 24 , wherein the polypeptide:
 a) is a therapeutically active or diagnostic polypeptide;   b) is susceptible to clipping by proteases;   c) comprises at least one clipping site for matriptase;   d) is a glycopolypeptide; and/or   e) is selected from the group consisting of glycoproteins, antibodies, non-IgG proteins, Fc-fusion proteins, Fab fragments, protein complexes, peptidases, signal peptides, nanobodies, growth factors, hormones, cytokines, blood factors and enzymes.   
     
     
         30 . The isolated vertebrate cell of  claim 24 , wherein (i) the at least one heterologous polynucleotide encoding the polypeptide of interest operatively linked to a secretory leader sequence is integrated into the genome of said cell and wherein optionally, at least one heterologous polynucleotide encoding a selectable marker or reporter polypeptide is additionally integrated into the genome of said cell; and/or (ii) the at least one heterologous polynucleotide encoding the polypeptide of interest operatively linked to a secretory leader sequence is comprised in an expression cassette. 
     
     
         31 . The isolated vertebrate cell of  claim 24 , wherein the vertebrate cell is a mammalian cell. 
     
     
         32 . The isolated vertebrate cell of  claim 31 , wherein the mammalian cell is a rodent cell. 
     
     
         33 . A method for producing the vertebrate cell of  claim 24 , comprising altering a vertebrate cell to impair the effect of matriptase by reducing or eliminating functional expression of the matriptase gene in said cell by gene knock-out, gene mutation, gene deletion, gene silencing or a combination thereof, and introducing a polynucleotide encoding a polypeptide of interest operatively linked to a secretory leader sequence, wherein said polypeptide of interest is secreted by the vertebrate cell. 
     
     
         34 . A method for recombinantly producing a polypeptide of interest, comprising
 (a) culturing vertebrate cell of  claim 24  under conditions that allow for the expression and secretion of the polypeptide of interest into the cell culture medium;   (b) isolating the polypeptide of interest from the cell culture medium; and   (c) optionally processing the isolated polypeptide of interest.   
     
     
         35 . A method for selecting a host cell which recombinantly expresses a polypeptide of interest, comprising
 (a) providing the vertebrate cell of  claim 24  as a host cell; and   (b) selecting one or more host cells expressing the polypeptide of interest;   wherein optionally step (a) comprises transfecting vertebrate cells in which the function of the endogenous protease matriptase is impaired with at least one polynucleotide encoding a polypeptide of interest operatively linked to a secretory leader sequence to provide the vertebrate host cells.   
     
     
         36 . The method of  claim 35 , having one or more of the following characteristics:
 a) said vertebrate cell provided in step (a) additionally comprises at least one heterologous polynucleotide encoding a selectable marker and step (b) comprises culturing said plurality of host cells under conditions selective for the selectable marker;   b) polynucleotides are introduced into the vertebrate cells by transfecting one or more expression vectors;   c) step (b) comprises one or multiple selection steps; and/or   d) step (b) comprises performing a flow cytometry-based selection.   
     
     
         37 . A method for selecting a vertebrate cell for recombinant production of a secreted polypeptide of interest, comprising
 (i) analyzing if endogenous protease matriptase is functionally expressed in a vertebrate cell,   (ii) selecting a vertebrate cell in which the effect of said endogenous protease matriptase is impaired by reduction or elimination of functional expression of the matriptase gene by gene knock-out, gene mutation, gene deletion, gene silencing or a combination thereof for recombinant production of the secreted polypeptide of interest, and   (iii) introducing a polynucleotide encoding the polypeptide of interest operatively linked to a secretory leader sequence into the vertebrate cell,   wherein step (iii) can occur before or after steps (i) and (ii), and wherein the polypeptide of interest is secreted by the vertebrate cell.   
     
     
         38 . The method of  claim 34 , wherein the genome of said vertebrate cell is altered to impair the function of the endogenous protease matriptase, and/or wherein at least one or all copies of the matriptase gene are deleted or functionally inactivated. 
     
     
         39 . The method of  claim 34 , wherein said vertebrate cells are provided as a cell clone or cell line. 
     
     
         40 . The method of  claim 34 , wherein the polypeptide:
 a) is a therapeutically active or diagnostic polypeptide;   b) is susceptible to clipping by proteases;   c) comprises at least one clipping site for matriptase;   d) is a glycopolypeptide; and/or   e) is selected from the group consisting of glycoproteins, antibodies, non-IgG proteins, Fc-fusion proteins, Fab fragments, protein complexes, peptidases, signal peptides, nanobodies, growth factors, hormones, cytokines, blood factors and enzymes.   
     
     
         41 . The method of  claim 34 , wherein (i) at least one heterologous polynucleotide encoding the polypeptide of interest operatively linked to a secretory leader sequence is integrated into the genome of said vertebrate cell and wherein optionally, at least one heterologous polynucleotide encoding a selectable marker or reporter polypeptide is additionally integrated into the genome of said cell; and/or (ii) said vertebrate cell comprises at least one heterologous polynucleotide encoding the polypeptide of interest operatively linked to a secretory leader sequence comprised in an expression cassette. 
     
     
         42 . The method of  claim 34 , wherein
 (a) said vertebrate cell comprises one or more mutations in at least one copy or all copies of the matriptase gene to provide a non- or less-functional expression product; or   (b) said vertebrate cell comprises one or more mutations in the promoter, in the 5′UTR, the 3′UTR and/or other regulatory elements of the matriptase gene.   
     
     
         43 . The method of  claim 42 , wherein said one or more mutations are comprised in a coding region of the matriptase gene and result in a non- or less functional expression product, wherein optionally the one or more mutations are comprised in a polynucleotide sequence of exon 2 of the matriptase gene or wherein the one or more mutations are comprised in a polynucleotide sequence of the matriptase gene that encodes at least part of the catalytic domain of matriptase whereby a non- or less functional expression product is obtained. 
     
     
         44 . The method of  claim 37 , wherein
 (i) the genome of said selected vertebrate cell is altered to impair the function of said endogenous protease matriptase, and/or wherein at least one or all copies of said matriptase gene are deleted or functionally inactivated;   (ii) said vertebrate cell is provided as a cell clone or cell line;   (iii) at least one heterologous polynucleotide encoding the polypeptide of interest operatively linked to a secretory leader sequence is integrated into the genome of said vertebrate cell and wherein optionally, at least one heterologous polynucleotide encoding a selectable marker or reporter polypeptide is additionally integrated into the genome of said vertebrate cell; and/or   (iv) said vertebrate cell comprises at least one heterologous polynucleotide encoding the polypeptide of interest operatively linked to a secretory leader sequence comprised in an expression cassette.   
     
     
         45 . The method of  claim 37 , wherein
 (a) said vertebrate cell comprises one or more mutations in at least one copy or all copies of the matriptase gene to provide a non- or less functional expression product; or   (b) said vertebrate cell comprises one or more mutations in the promoter, in the 5′UTR, the 3′UTR and/or other regulatory elements of the matriptase gene.   
     
     
         46 . The method of  claim 45 , wherein said one or more mutations are comprised in a coding region of the matriptase gene and result in a non- or less functional expression product, wherein optionally the one or more mutations are comprised in a polynucleotide sequence of exon 2 of the matriptase gene or wherein the one or more mutations are comprised in a polynucleotide sequence of the matriptase gene that encodes at least part of the catalytic domain of matriptase whereby a non- or less functional expression product is obtained. 
     
     
         47 . An isolated human cell suitable for recombinant expression of a polypeptide of interest, wherein the human cell is altered to impair the effect of matriptase and comprises at least one heterologous polynucleotide encoding a polypeptide of interest, wherein the vertebrate cell secretes the polypeptide of interest. 
     
     
         48 . The isolated vertebrate cell according to  claim 47 , wherein the unaltered endogenous matriptase shares at least 80% identity to a matriptase reference protein comprising one or more of the amino acid sequences set forth in SEQ ID NOs: 2 to 5 and has the same proteolytic activity as said reference matriptase protein.