Mammalian cells for producing a secreted protein

Abstract

The invention relates to the field of cell culture technology. It concerns the knockdown, using RNA interference, or gene knockout, of activating transcription factor 6 beta (ATF6B), or the combination of ceramide synthase 2 (CERS2) and TBC1 domain family member 20 (TBC1D20) proteins, which play central roles in the cellular secretion pathway. This downregulation leads to improved secretion of biopharmaceutically relevant products produced in mammalian cells. The invention specifically relates to mammalian cells having enhanced secretion of a recombinant therapeutic protein compared to a control cell, a method of producing said mammalian cell, a method for the production of a recombinant secreted therapeutic protein and the use of said mammalian cell for increasing the yield of a recombinant secreted therapeutic protein.

1 . A mammalian cell engineered to have enhanced secretion of a recombinant therapeutic protein compared to a non-engineered control cell, said engineered mammalian cell comprising
 (a) reduced expression of the host cell proteins TBC1 domain family member 20 (TBC1D20) and ceramide synthase 2 (CERS2); or   (b) reduced expression of the host cell protein activating transcription factor 6 beta (ATF6B), wherein the engineered mammalian cell further comprises one or more expression cassette(s) encoding a recombinant secreted therapeutic protein;   wherein the engineered mammalian cell is not an engineered Chinese Hamster Ovary (CHO) cell.   
     
     
         2 . A mammalian cell engineered to have enhanced secretion of a recombinant therapeutic protein compared to a non-engineered control cell, said engineered mammalian cell comprising reduced expression of the host cell proteins TBC1D20 and CERS2;
 wherein the engineered mammalian cell is not an engineered Chinese Hamster Ovary (CHO) cell.   
     
     
         3 . The engineered mammalian cell of  claim 2 , further comprising one or more expression cassette(s) encoding a recombinant secreted therapeutic protein. 
     
     
         4 . The engineered mammalian cell of  claim 1 , wherein:
 (a) the gene(s) encoding the host cell protein(s) comprise(s) a genetic modification that inhibits expression of said host cell protein(s), or   (b) the engineered mammalian cell comprises (an) RNA oligonucleotide(s) that inhibit(s) inhibits expression of the gene(s) encoding said host cell protein(s) by RNA-interference, and   wherein the protein expression of TBC1D20 and CERS2 or the protein expression of ATF6B in the engineered mammalian cell is reduced compared to the same mammalian cell not containing said genetic modification(s) or RNA oligonucleotide(s).   
     
     
         5 . A method of producing an engineered mammalian cell with enhanced secretion of a recombinant therapeutic protein, said method comprising
 (a) reducing expression of the host cell proteins TBC1D20 and CERS2, or of the host cell protein ATF6B, in the mammalian cell by introducing   (i) a genetic modification into the gene(s) encoding the host cell protein(s) that inhibits expression of said host cell protein(s), or   (ii) (an) RNA oligonucleotide(s) into the mammalian cell that inhibit(s) expression of the gene(s) encoding said host cell protein(s) by RNA-interference, and   (b) introducing one or more gene(s) encoding a recombinant secreted therapeutic protein;   wherein the engineered mammalian cell is not an engineered Chinese Hamster Ovary (CHO) cell.   
     
     
         6 . The method of  claim 5 , further comprising the following steps:
 (c) selecting cells with enhanced secretion of the recombinant therapeutic protein; and   (d) optionally culturing the cells obtained in step (c) under conditions which allow expression of one or more gene(s) encoding a recombinant secreted therapeutic protein.   
     
     
         7 . The method of  claim 5 , wherein the protein expression of TBC1D20 and CERS2 or the protein expression of ATF6B in the engineered mammalian cell is reduced compared to the same mammalian cell not containing said genetic modification(s) or RNA oligonucleotide(s). 
     
     
         8 . The engineered mammalian cell of  claim 4 , wherein the engineered mammalian cell comprises (an) RNA oligonucleotide(s) that inhibit(s) expression of the gene(s) encoding said host cell protein(s) by RNA-interference and wherein the RNA-interference is mediated by small hairpin RNA (shRNA) or short interfering RNA (siRNA). 
     
     
         9 . The engineered mammalian cell of  claim 8 , wherein the mammalian cell is transfected with one or more expression vector(s) comprising one or more nucleotide sequence(s) encoding said siRNA(s) or shRNA(s), and optionally wherein the mammalian cell is stably transfected with one or more expression vector(s) comprising the nucleotide sequence(s) encoding said siRNA(s) or shRNA(s). 
     
     
         10 . The engineered mammalian cell of  claim 8 , wherein
 (i) the siRNA is   (a) siTbc1D20 #1 (SEQ ID NO: 7) or siCerS2 #1 (SEQ ID NO: 8), or a combination thereof; or   (b) one or more of siAtf6b #1 (SEQ ID NO: 9), siAtf6b #2 (SEQ ID NO: 10), and siAtf6b #3 (SEQ ID NO: 11); or   (ii) the shRNA comprises   (a) shTbc1D20 #1 (SEQ ID NO: 12) or one or more of shCerS2 #1 (SEQ ID NO: 13) and shCerS2 #2 (SEQ ID NO: 14), or a combination thereof; or   (b) one or more of shAtf6b #1 (SEQ ID NO: 15) and shAtf6b #2 (SEQ ID NO: 37).   
     
     
         11 . The engineered mammalian cell of  claim 4 , wherein the gene(s) encoding the host cell protein(s) TBC1D20 and CERS2, or ATF6B, comprise(s) a genetic modification that inhibits expression of said host cell protein(s) and wherein the genetic modification in the gene(s) encoding the host cell protein(s) TBC1D20 and CERS2, or ATF6B, is independently
 (a) a gene deletion; or   (b) a mutation in the gene that inhibits expression of the host cell protein, wherein said mutation is in the coding region of the gene and/or the promoter or regulatory region of the gene.   
     
     
         12 . The engineered mammalian cell of  claim 1 , wherein:
 (a) the engineered mammalian cell comprises reduced expression of the host cell proteins TBC1D20 and CERS2 and wherein the host cell protein TBC1D20 has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 4 and the host cell protein CERS2 has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 5; or   (b) the engineered mammalian cell comprises reduced expression of the host cell protein ATF6B and wherein the host cell protein ATF6B has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 6.   
     
     
         13 . The engineered mammalian cell of  claim 1 , wherein
 (i) the said recombinant secreted therapeutic protein is   (a) an antibody, preferably a monoclonal antibody, a bi-specific antibody or a fragment thereof, or   (b) an Fc-fusion protein; or   (ii) the cell is a rodent or a human cell, preferably a CHO cell.   
     
     
         14 . A method for the production of a recombinant secreted therapeutic protein in a mammalian cell comprising
 (a) providing an engineered mammalian cell of  claim 1 , wherein the cell is transfected with a recombinant secreted therapeutic protein,   (b) culturing the mammalian cell of step (a) in a cell culture medium at conditions allowing production of the recombinant secreted therapeutic protein,   (c) harvesting the recombinant secreted therapeutic protein, and optionally   (d) purifying the recombinant secreted therapeutic protein.   
     
     
         15 . Use of the engineered mammalian cell of  claim 1  for increasing the yield of a recombinant secreted therapeutic protein. 
     
     
         16 . The method of  claim 5 , wherein the engineered mammalian cell comprises (an) RNA oligonucleotide(s) that inhibit(s) expression of the gene(s) encoding said host cell protein(s) by RNA-interference and wherein the RNA-interference is mediated by small hairpin RNA (shRNA) or short interfering RNA (siRNA). 
     
     
         17 . The method of  claim 16 , wherein:
 (A) the mammalian cell is transfected with one or more expression vector(s) comprising one or more nucleotide sequence(s) encoding said siRNA(s) or shRNA(s), and optionally wherein the mammalian cell is stably transfected with one or more expression vector(s) comprising the nucleotide sequence(s) encoding said siRNA(s) or shRNA(s); and/or   (B) wherein:
 (i) the siRNA is
 (a) siTbc1D20 #1 (SEQ ID NO: 7) or siCerS2 #1 (SEQ ID NO: 8), or a combination thereof; or 
 (b) one or more of siAtf6b #1 (SEQ ID NO: 9), siAtf6b #2 (SEQ ID NO: 10), and siAtf6b #3 (SEQ ID NO: 11); or 
 
 (ii) the shRNA comprises
 (a) shTbc1D20 #1 (SEQ ID NO: 12) or one or more of shCerS2 #1 (SEQ ID NO: 13) and shCerS2 #2 (SEQ ID NO: 14), or a combination thereof; or 
 (b) one or more of shAtf6b #1 (SEQ ID NO: 15) and shAtf6b #2 (SEQ ID NO: 
 
   37).   
     
     
         18 . The method of  claim 5 , wherein the gene(s) encoding the host cell protein(s) TBC1D20 and CERS2, or ATF6B, comprise(s) a genetic modification that inhibits expression of said host cell protein(s) and wherein the genetic modification in the gene(s) encoding the host cell protein(s) TBC1D20 and CERS2, or ATF6B, is independently
 (a) a gene deletion; or   (b) a mutation in the gene that inhibits expression of the host cell protein, wherein said mutation is in the coding region of the gene and/or the promoter or regulatory region of the gene.   
     
     
         19 . The engineered mammalian cell of  claim 4 , wherein:
 (a) the engineered mammalian cell comprises reduced expression of the host cell proteins TBC1D20 and CERS2 and wherein the host cell protein TBC1D20 has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 4 and the host cell protein CERS2 has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 5; or   (b) the engineered mammalian cell comprises reduced expression of the host cell protein ATF6B and wherein the host cell protein ATF6B has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 6.   
     
     
         20 . The method of  claim 5 , wherein:
 (a) the engineered mammalian cell comprises reduced expression of the host cell proteins TBC1D20 and CERS2 and wherein the host cell protein TBC1D20 has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 4 and the host cell protein CERS2 has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 5; or   (b) the engineered mammalian cell comprises reduced expression of the host cell protein ATF6B and wherein the host cell protein ATF6B has sequence identity of at least 80% to the amino acid sequence of SEQ ID NO: 6.