title: Enhancement of DNA uptake in FUT8-deleted CHO cells for transient production of afucosylated antibodies.
abstract: Removal of the core alpha1,6 fucose from the glycans in the Fc region of IgG1 antibodies has been demonstrated to improve antibody-dependent cellular cytotoxicity (ADCC) activity. In order to produce afucosylated antibodies using transient transfection, a FUT8 knockout (FUT8KO) cell line was generated in a CHO host cell line using the zinc finger nuclease technology. Transient transfection of DNA into mammalian cells using the cationic polymer, polyethylenimine (PEI), is commonly used for rapid generation of recombinant proteins. FUT8KO cells evaluated in PEI transfections yielded lower titers than parental CHO WT cells. FACS and HPLC analyses revealed that the FUT8KO cells had lower cell surface heparan sulfate (HS) levels than CHO WT. Removal of cell surface HS resulted in reduced uptake of PEI-transfected DNA (PEI:DNA) and lower transfection titers suggesting that PEI:DNA relies on HS for binding and cellular entry. The absence of cell surface HS did not severely impact transfections performed with cationic liposomes. We undertook two approaches to improve transient production of afucosylated antibodies. First, we evaluated transfection of FUT8KO cells with cationic liposomes, which were observed to be less dependent on HS levels for uptake. Transfection of FUT8KO cells using the cationic liposome, DMRIE-C, produced similar titers to CHO WT in both shake flask and large-scale 10 L bioreactors. The second approach was to engineer a cell line overexpressing exostosin-1 (EXT1), an enzyme responsible for HS chain elongation, to increase HS content. EXT1-FUT8KO and CHO WT cells produced comparable levels of antibody from PEI transfections.
authors.affiliation: Department of Early Stage Cell Culture, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA. wong.athena@gene.com
authors.forename: Athena W
authors.initials: AW
authors.lastname: Wong
authors.forename: Tomasz K
authors.initials: TK
authors.lastname: Baginski
authors.forename: Dorothea E
authors.initials: DE
authors.lastname: Reilly
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D016466:CHO Cells; D018929:Cell Culture Techniques; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D005647:Fucosyltransferases / Q000172:deficiency; D015870:Gene Expression; D055786:Gene Knockout Techniques; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D004247:DNA; D005647:Fucosyltransferases