Compositions and methods for reducing fucosylation of glycoproteins in insect cells and methods of use thereof for production of recombinant glycoproteins

Abstract

Compositions for reducing fucosylation of glycoproteins in insect cells are provided. Also disclosed are methods of use of such compositions for the production of recombinant humanized proteins.

What is claimed is: 
     
         1 . A recombinant baculovirus expression vector for transient expression of GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) in an insect cell, said vector comprising the following operably linked components,
 i) an expression control sequence functional early in infection operably linked to a codon optimized RMD encoding nucleic acid; and   ii) an insertion site suitable for insertion of one or more nucleic acids encoding at least one heterologous protein of interest.   
     
     
         2 . The recombinant baculovirus expression vector of  claim 1 , which is AcRMD. 
     
     
         3 . The recombinant baculovirus expression vector of  claim 1  or  claim 2  comprising a nucleic acid sequence encoding a heterologous protein of interest operably linked to a promoter active later in infection. inserted at said insertion site. 
     
     
         4 . The recombinant baculovirus expression vector of  claim 1 , wherein said expression control sequence is selected from the group consisting of a constitutive promoter and an inducible promoter. 
     
     
         5 . The recombinant baculovirus expression vector of  claim 4 , wherein said constitutive promoter is a baculovirus immediate early promoter selected from the group consisting of ie1, ie2, ie0, et1, and gp64,insect actin, tubulin, a ubiquitin promoter; RSV promoter, copia, gypsy promoter and a cytomegalovirus IE promoter. 
     
     
         6 . The recombinant baculovirus expression vector of  claim 4 , wherein said inducible promoter is selected from the group consisting of baculovirus delayed early, late, and very late promoters, an hsp70 promoter, a metallothionein promoter and a tetracycline-regulated promoter. 
     
     
         7 . The recombinant baculovirus expression vector of  claim 3 , wherein said nucleic acid encoding said at least one heterologous protein of interest comprises a promoter selected from the group consisting of a promoter from baculovirus delayed early, late, and very late promoters. 
     
     
         8 . The recombinant baculovirus expression vector of  claim 4 , wherein said expression control sequence comprises a promoter and an enhancer element that increases activity of said promoter. 
     
     
         9 . The recombinant baculovirus expression vector of  claim 3 , wherein said protein of interest is a therapeutic protein. 
     
     
         10 . The recombinant baculovirus expression vector of  claim 9 , wherein said protein of interest is selected from the group consisting of an antibody, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, and a blood clotting factor. 
     
     
         11 . An infected insect cell comprising the recombinant baculoviral vector of  claim 1  or  claim 3 , selected from the group consisting of Sf9, Sf21, expresSF+®, Tn368, High Five®, Tni PRO®, Ea4, Ao38, BmN, S2, and S2R+. 
     
     
         12 . A method for producing at least one molecule of interest lacking fucose, comprising:
 a) providing insect cells;   b) introducing a baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one heterologous protein of interest driven by an promoter active later in infection, thereby producing non-fucosylated proteins wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector;   c) incubating under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose; and   d) isolating said at least one protein of interest.   
     
     
         13 . The method of  claim 12 , wherein said RMD enzyme and said protein of interest are encoded by a single recombinant baculoviral vector and expressed sequentially at earlier and later times of infection. 
     
     
         14 . The method of  claim 12 , wherein said nucleic acid encoding said enzyme and said protein of interest are on separate baculovirus vectors and expressed sequentially at earlier and later times of infection. 
     
     
         15 . The method of  claim 12 , wherein said protein of interest is a therapeutic protein. 
     
     
         16 . The method of  claim 15 , wherein said therapeutic protein is selected from the group consisting of an antibody, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, and a blood clotting factor. 
     
     
         17 . The method of  claim 16 , wherein said therapeutic protein is an antibody. 
     
     
         18 . A kit for the production of at least one protein of interest lacking fucose or with a reduced amount of fucose comprising at least one recombinant baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression early after infection, thereby stabilizing inhibition of fucosylation, and an insertion site for at least one additional nucleic acid molecule encoding at least one protein of interest operably driven by a control sequence for expression later in infection for production of non-fucosylated proteins of interest. 
     
     
         19 . The kit of  claim 18 , further comprising insect cells. 
     
     
         20 . The kit of  claim 18 , further comprising a second baculoviral vector comprising a promoter suitable to drive expression of said protein of interest later in in infection and an insertion site for insertion of a nucleic acid encoding said protein of interest. 21. The kit of  claim 18 , wherein said protein of interest is selected from the group consisting of an antibody, cytokine, blood clotting factor, anticoagulant, viral antigen, enzyme, receptor, vaccine, subunit vaccine, and hormone. 
     
     
         22 . A method for production of a non-fucosylated protein in insect larvae, comprising
 a) providing insect larvae;   b) introducing a baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one protein of interest driven by an expression control sequence active later in infection, thereby producing non-fucosylated proteins wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector;   c) incubating under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose; and   d) isolating said protein of interest.   
     
     
         23 . The recombinant baculovirus vector of  claim 8 , wherein said enhancer is hr5.