Cell lines and methods for producing proteins

Abstract

The present invention relates to the isolation and enrichment of avian oviduct tumor cells to obtain sustainable cell lines that can be passaged multiple times in cell culture and can be used for long-term production of heterologous polypeptides at higher yields. The methods of the instant invention involve the manipulation and/or propagation of oviduct tumor cells derived from either wild-type or transgenic avians.

1 . An isolated avian oviduct tumor cell line comprising a nucleic acid encoding a heterologous protein, wherein the cell line is sustainable in culture for at least three or more passages.  
     
     
         2 . The cell line of  claim 1  wherein the cell line is sustainable in culture for at least ten or more passages.  
     
     
         3 . The cell line of  claim 1  wherein the cell line is sustainable in culture for at least twenty or more passages.  
     
     
         4 . The cell line of  claim 1  wherein the nucleic acid is operably linked to a promoter.  
     
     
         5 . The cell line of  claim 4  wherein the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a MDOT promoter and a rous-sarcoma virus (RSV) promoter.  
     
     
         6 . The cell line of  claim 1  wherein the nucleic acid is operably linked to an oviduct-specific promoter.  
     
     
         7 . The cell line of  claim 6  wherein the oviduct-specific promoter is selected from an ovomucoid promoter, an ovalbumin promoter, and a lysozyme promoter.  
     
     
         8 . The cell line of  claim 1  wherein the heterologous protein is a pharmaceutical composition.  
     
     
         9 . The cell line of  claim 1  wherein the avian is selected from the group consisting of a chicken, turkey, duck, goose, quail, pheasant, parrot, finch, hawk, crow or ratite, ostrich, emu and cassowary.  
     
     
         10 . The cell line of  claim 1  wherein the avian is a chicken.  
     
     
         11 . The cell line of  claim 1  isolated from a transgenic avian that expresses the heterologous protein in oviduct cells.  
     
     
         12 . The cell line of  claim 1  wherein the tumor cell line is an epithelial or mesenchymal tumor.  
     
     
         13 . The cell line of  claim 1  wherein the tumor is an epithelial adenoma or adenocarcinoma.  
     
     
         14 . The cell line of  claim 1  wherein the tumor is an epithelial adenoma.  
     
     
         15 . A composition comprising the cell line of  claim 1  and a culture medium.  
     
     
         16 . The composition of  claim 15  wherein the culture medium comprises a heterologous protein expressed by the cell line.  
     
     
         17 . An immortal cell line derived from a tubular gland cell comprising a nucleic acid encoding a heterologous protein.  
     
     
         18 . The cell line of  claim 17  wherein the nucleic acid is operably linked to a promoter.  
     
     
         19 . The cell line of  claim 17  wherein the nucleic acid is operably linked to an oviduct-specific promoter.  
     
     
         20 . The cell line of  claim 17  wherein the promoter is selected from an ovomucoid promoter, an ovalbumin promoter, and a lysozyme promoter.  
     
     
         21 . The cell line of  claim 17  wherein the protein is a pharmaceutical composition.  
     
     
         22 . The cell line of  claim 17  wherein the protein is a cytokine.  
     
     
         23 . The cell line of  claim 17  wherein the protein is an antibody.  
     
     
         24 . The cell line of  claim 17  wherein the protein is selected from the group consisting of a mammalian monoclonal antibody, a fusion protein, a growth factor, an enzyme, a structural protein, interferon, granulocyte-colony stimulating factor, and erythropoietin.  
     
     
         25 . The cell line of  claim 17  isolated from a transgenic avian that expresses the heterologous protein in oviduct cells.  
     
     
         26 . The cell line of  claim 17  wherein the tumor cell line is an epithelial or mesenchymal tumor.  
     
     
         27 . The cell line of  claim 17  wherein the tumor is an epithelial adenoma or adenocarcinoma.  
     
     
         28 . The cell line of  claim 17  wherein the tumor is an epithelial adenoma.  
     
     
         29 . A composition comprising the cell line of  claim 17  and a culture medium.  
     
     
         30 . The composition of  claim 29  wherein the culture medium comprises a heterologous protein expressed by the cell line.  
     
     
         31 . A method of producing a heterologous protein comprising, 
 contacting an avian oviduct tumor cell with a nucleic acid encoding a heterologous protein under conditions suitable for protein expression, wherein the tumor cell is sustainable in culture for at least three or more passages; and    isolating the heterologous protein.    
     
     
         32 . The method of  claim 31  wherein the nucleic acid is transfected into the cell in vitro.  
     
     
         33 . The method of  claim 31  wherein the nucleic acid is contacted with the cell in vivo.  
     
     
         34 . The method of  claim 31  wherein the nucleic acid comprises a selectable marker, and wherein the method further comprises selecting for tumor cells that express the marker.  
     
     
         35 . The method of  claim 31  further comprising enriching for tumor cells that express the heterologous protein.  
     
     
         36 . The method of  claim 35  wherein the enriching comprises selecting for cells that express gicerin.  
     
     
         37 . The method of  claim 36  wherein the selection is performed using a fluorescently labeled antibody that binds gicerin.  
     
     
         38 . The method of  claim 31  further comprising enriching for tumor cells that bind neurite outgrowth factor (NOF).  
     
     
         39 . The method of  claim 31  wherein the tumor is an epithelial adenoma.  
     
     
         40 . The method of  claim 31  wherein the avian is a chicken.  
     
     
         41 . The method of  claim 31  wherein the transfection is performed by microinjection, electroporation or lipofection.  
     
     
         42 . The method of  claim 31  wherein the tumor cell is sustainable in culture for at least ten or more passages.  
     
     
         43 . The method of  claim 31  wherein the tumor cell is sustainable in culture for at least twenty or more passages.  
     
     
         44 . The method of  claim 31  wherein the protein is a pharmaceutical composition.  
     
     
         45 . The method of  claim 31  wherein the protein is a cytokine.  
     
     
         46 . The method of  claim 31  wherein the protein is an antibody.  
     
     
         47 . The method of  claim 31  wherein the protein is selected from the group consisting of a mammalian monoclonal antibody, a fusion protein, a growth factor, an enzyme, a structural protein, interferon, granulocyte-colony stimulating factor, and erythropoietin.  
     
     
         48 . A method of producing a heterologous protein comprising: 
 isolating oviduct tumor cells that expresses the protein, wherein the cell is sustainable in culture for at least three or more passages;    culturing the tumor cells under conditions suitable for expression of the protein; and    isolating the protein.    
     
     
         49 . The method of  claim 48  wherein the tumor cell is sustainable in culture for at least ten or more passages.  
     
     
         50 . The method of  claim 48  wherein the tumor cell is sustainable in culture for at least twenty or more passages.  
     
     
         51 . The method of  claim 48  wherein the avian is a chicken.  
     
     
         52 . The method of  claim 48  wherein the protein is a pharmaceutical composition.