Universal immunomodulatory cytokine-expressing bystander cell line and related compositions and methods of manufacture and use

Abstract

The present invention provides a universal immunomodulatory cytokine-expressing bystander cell line, a composition comprising such a cell line and a cancer antigen, a method of making such a cell line, and a method of using such a composition.

What is claimed is:  
     
         1 . A universal bystander cell line, which: 
 (i) is a human cell line,    (ii) naturally lacks major histocompatibility class I (MHC-I) antigens and major histocompatibility class II (MHC-II) antigens or is modified so that it lacks MEC-I antigens and MHC-II antigens, and    (iii) is modified by introduction of a nucleic acid molecule comprising a nucleic acid sequence encoding granulocyte macrophage-colony stimulating factor (GM-CSF) operably linked to a promoter,    wherein said universal bystander cell line expresses at least about 500 ng GM-CSF/10 6  cells/24 hours.    
     
     
         2 . The universal bystander cell line of  claim 1 , wherein said human cell line is characterized by the absence of B-lymphocyte markers of immunoglobulin, an Epstein-Barr virus (EBV) genome and an associated nuclear antigen, and receptors for EBV.  
     
     
         3 . The universal bystander cell line of  claim 1 , wherein said human cell line is derived from a blast crisis of chronic myeloid leukemia.  
     
     
         4 . The universal bystander cell line of  claim 1 , wherein said human cell line is K562.  
     
     
         5 . The universal bystander cell line of  claim 1 , which expresses at least about 1,000 ng GM-CSF/10 6  cells/24 hours.  
     
     
         6 . The universal bystander cell line of  claim 1 , which grows in defined medium.  
     
     
         7 . The universal bystander cell line of  claim 1 , wherein said promoter is a cytomegalovirus promoter.  
     
     
         8 . The universal bystander cell line of  claim 4 , which expresses at least about 1,000 ng GM-CSF/10 6  cells/24 hours.  
     
     
         9 . The universal bystander cell line of  claim 4 , which grows in defined medium.  
     
     
         10 . The universal bystander cell line of  claim 4 , wherein said promoter is a cytomegalovirus promoter.  
     
     
         11 . The universal bystander cell line of  claim 1 , wherein said nucleic acid molecule further comprises a nucleic acid sequence encoding hygromycin resistance operably linked to a promoter and said universal bystander cell line is selected by growth in a culture medium comprising at least about 400 μg/ml hygromycin.  
     
     
         12 . The universal bystander cell line of  claim 11 , wherein said universal bystander cell line is selected by growth in a culture medium comprising at least about 1,000 μg/ml hygromycin.  
     
     
         13 . The universal bystander cell line of  claim 4 , wherein said nucleic acid molecule further comprises a nucleic acid sequence encoding hygromycin resistance operably linked to a promoter and said universal bystander cell line is selected by growth in a culture medium comprising at least about 400 μg/ml hygromycin.  
     
     
         14 . The universal bystander cell line of  claim 13 , wherein said universal bystander cell line is selected by growth in a culture medium comprising at least about 1,000 μg/ml hygromycin.  
     
     
         15 . A composition comprising: 
 (a) a universal bystander cell line, which 
 (i) is a human cell line, (ii) naturally lacks MHC-I antigens and MHC-II antigens or is modified so that it lacks MHC-I antigens and MHC-II antigens, and (iii) is modified by introduction of a nucleic acid molecule comprising a nucleic acid sequence encoding an immunomodulatory cytokine operably linked to a promoter, and  
   (b) a cancer antigen.    
     
     
         16 . The composition of  claim 15 , wherein said immunomodulatory cytokine is interleukin-2 (IL-2).  
     
     
         17 . A composition comprising the universal bystander cell line of  claim 1  and a cancer antigen.  
     
     
         18 . A composition comprising the universal bystander cell line of  claim 2  and a cancer antigen.  
     
     
         19 . A composition comprising the universal bystander cell line of  claim 4  and a cancer antigen.  
     
     
         20 . A composition comprising the universal bystander cell line of  claim 5  and a cancer antigen.  
     
     
         21 . A composition comprising the universal bystander cell line of  claim 8  and a cancer antigen.  
     
     
         22 . A method of making a universal GM-CSF-expressing bystander cell line, which method comprises: 
 (i) obtaining a human cell line that lacks MHC-I antigens and MHC-II antigens;    (ii) modifying said human cell line by introducing into said human cell line a nucleic acid molecule comprising a nucleic acid sequence encoding GM-CSF operably linked to a promoter and a nucleic acid sequence encoding a selectable marker operably linked to a promoter; and    (iii) using the selectable marker to isolate cells that produce at least about 500 ng of said GM-CSF/10 6  cells/24 hours.    
     
     
         23 . The method of  claim 22 , wherein said selectable marker is hygromycin resistance.  
     
     
         24 . The method of  claim 23 , wherein the modified human cell line is cultured in culture medium comprising at least about 400 μg hygromycin/ml culture medium.  
     
     
         25 . The method of  claim 24 , wherein the modified human cell line is subsequently cultured in culture medium comprising at least about 1,000 μg hygromycin/ml culture medium.  
     
     
         26 . The method of  claim 24 , wherein said culture medium is defined.  
     
     
         27 . The method of  claim 25 , wherein said culture medium is defined.  
     
     
         28 . The method of  claim 22 , wherein the promoter to which the nucleic acid sequence encoding GM-CSF is operably linked is a cytomegalovirus promoter.  
     
     
         29 . A method of making a universal GM-CSF-expressing bystander cell line, which method comprises: 
 (i) obtaining a human cell line;    (ii) modifying said human cell line so that it lacks MHC-I antigens and MHC-II antigens;    (iii) further modifying said human cell line by introducing into said human cell line a nucleic acid molecule comprising a nucleic acid sequence encoding GM-CSF operably linked to a promoter and a nucleic acid sequence encoding a selectable marker operably linked to a promoter; and    (iv) using the selectable marker to isolate cells that produce at least about 500 ng of GM-CSF/10 6  cells/24 hours.    
     
     
         30 . The method of  claim 29 , wherein said selectable marker is hygromycin resistance.  
     
     
         31 . The method of  claim 30 , wherein the modified human cell line is cultured in culture medium comprising at least about 400 μg hygromycin/ml culture medium.  
     
     
         32 . The method of  claim 31 , wherein the modified human cell line is subsequently cultured in culture medium comprising at least about 1,000 μg hygromycin/ml culture medium.  
     
     
         33 . The method of  claim 31 , wherein said culture medium is defined.  
     
     
         34 . The method of  claim 32 , wherein said culture medium is defined.  
     
     
         35 . The method of  claim 29 , wherein the promoter to which the nucleic acid sequence encoding GM-CSF is operably linked is a cytomegalovirus promoter.  
     
     
         36 . A method of stimulating an immune response to a cancer in a human patient, which method comprises administering to said patient the composition of  claim 15 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated, 
 whereupon administration of said composition, an immune response to said cancer is stimulated.    
     
     
         37 . The method of  claim 36 , wherein said cancer antigen is a cell of said cancer.  
     
     
         38 . A method of stimulating an immune response to a cancer in a human patient, which method comprises administering to said patient the composition of  claim 16 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated, 
 whereupon administration of said composition, an immune response to said cancer is stimulated.    
     
     
         39 . The method of  claim 38 , wherein said cancer antigen is a cell of said cancer.  
     
     
         40 . A method of stimulating an immune response to a cancer in a human patient, which method comprises administering to said patient the composition of  claim 17 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated, 
 whereupon administration of said composition, an immune response to said cancer is stimulated.    
     
     
         41 . The method of  claim 40 , wherein said cancer antigen is a cell of said cancer.  
     
     
         42 . A method of stimulating an immune response to a cancer in a human patient, which method comprises administering to said patient the composition of  claim 19 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated, 
 whereupon administration of said composition, an immune response to said cancer is stimulated.    
     
     
         43 . The method of  claim 42 , wherein said cancer antigen is a cell of said cancer.  
     
     
         44 . A method of stimulating an immune response to a cancer in a human patient, which method comprises administering to said patient the composition of  claim 20 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated, 
 whereupon administration of said composition, an immune response to said cancer is stimulated.    
     
     
         45 . The method of  claim 44 , wherein said cancer antigen is a cell of said cancer.  
     
     
         46 . A method of stimulating an immune response to a cancer in a human patient, which method comprises administering to said patient the composition of  claim 21 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated, 
 whereupon administration of said composition, an immune response to said cancer is stimulated.    
     
     
         47 . The method of  claim 46 , wherein said cancer antigen is a cell of said cancer.  
     
     
         48 . In a method of cancer immunotherapy, the improvement comprising administering to a human patient having a cancer the composition of  claim 15 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated.  
     
     
         49 . In a method of cancer immunotherapy, the improvement comprising administering to a human patient having a cancer the composition of  claim 16 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated.  
     
     
         50 . In a method of cancer immunotherapy, the improvement comprising administering to a human patient having a cancer the composition of  claim 17 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated.  
     
     
         51 . In a method of cancer immunotherapy, the improvement comprising administering to a human patient having a cancer the composition of  claim 19 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated.  
     
     
         52 . In a method of cancer immunotherapy, the improvement comprising administering to a human patient having a cancer the composition of  claim 20 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated.  
     
     
         53 . In a method of cancer immunotherapy, the improvement comprising administering to a human patient having a cancer the composition of  claim 21 , wherein said cancer antigen is an antigen of said cancer and wherein said composition is irradiated.