O-glycan sialylated recombinant glycoproteins and cell lines for producing the same
The present invention relates to cell lines that are genetically modified to overexpress a β-galactoside α-2,3-sialyltransferase 1 (ST3Gal1), preferably human ST3Gal1, which can be used for the production of recombinant glycoproteins having highly or fully sialylated O-linked GalNAc glycans (GalNAc O-glycans), preferably core 1 GalNAc O-glycans, as well as to respective recombinant glycoproteins. Further, the present invention relates to respective methods of expressing recombinant glycoproteins, methods of increasing the degree of sialylation of recombinant glycoproteins, and methods of decreasing the micro-heterogeneity of GalNAc O-glycans. Finally, the present invention relates to respective uses of the above cell lines for the production of recombinant glycoproteins, for increasing the degree of sialylation of recombinant glycoproteins, and for decreasing the micro-heterogeneity of O-linked GalNAc glycans of recombinant glycoproteins.
1. A cell line that is genetically modified to overexpress a β-galactoside α-2,3-sialyltransferase 1 (ST3Gal1), which is further genetically modified to overexpress a β-galactoside α-2,3-sialyltransferase 4 (ST3Gal4), wherein said cell line can produce a glycoprotein comprising a sialylated GalNAc-O-glycan, said glycoprotein having an increased serum half-life as compared to a nonsialylated glycoprotein.
2. The cell line according to claim 1 , which is further genetically modified to overexpress a β-galactoside α-2,6-sialyltransferase 1 (ST6Gal1).
3. The cell line according to claim 1 , wherein the cell line comprises endogenous genes encoding ST3Gal1 and ST3Gal4, and optionally an endogenous gene encoding ST6Gal1, and further has at least one genetic element, selected from the group consisting of a promoter, an enhancing element, and a stabilizing element inserted into the genome in one or more position(s) suitable to cause overexpression of ST3Gal1 and ST4Gal4, and optionally ST6Gal1.
4. The cell line according to claim 1 , wherein the cell line comprises exogenous nucleic acids encoding ST3Gal1 and ST3Gal4, and optionally an exogenous nucleic acid encoding ST6Gal1.
5. The cell line according to claim 1 , wherein the cell line is a mammalian cell line.
6. The cell line according to claim 1 , wherein the cell line is derived from a cell line, selected from the group consisting of AGE.CR® cells, Vero cells, MDCK cells, BHK cells, CHO cells, HEK293 cells, HepG2 cells, Huh7 cells, AGE1.HN® cells, NC5T11 cells, Per.C6 cells, HMCLs cells, MM.1 cells, U266 cells, RPMI18226 cells, HKB11 cells, NM cells, NM-F9 cells, and CAP cells.
7. The cell line according to claim 1 , wherein the cell line is derived from human primary amniocytes comprising at least one nucleic acid encoding the gene products of the adenoviral E1 and pIX regions.
8. The cell line of claim 5 , wherein the mammalian cell line is a human cell line.