Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with humanN-glycosylation and improved pharmacokinetics

Abstract

N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4 and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.

Identification of novel molecular subtypes and a signature to predict prognosis and therapeutic response based on cuproptosis-related genes in prostate cancer

“…Studies have shown that FAM83D is strongly associated with cancer development, proliferation, invasion, and metastasis ( 78 , 79 ). Beta-1,4-N-acetylgalactosaminyltransferase 4 (B4GALNT4), as an N-acetylgalactosamine transferase, is involved in the post-translational regulation of genes through protein glycosylation modifications ( 80 ). B4GALNT4 is upregulated in various cancers, and its expression can enhance the malignant potential of cancers ( 81 ).…”

State-of-the-Art Approaches to Heterologous Expression of Bispecific Antibodies Targeting Solid Tumors

“…However, recently developed glycoengineered HEK293-F cell line eliminates terminal GalNAc glycosylation and increases sialylation due to the knockout of GalNAc transferases B4GALNT3 and B4GALNT4 and overexpression of sialyltransferases ST6GAL1 and ST3GAL6. This glycoengineered cell line is well-suited for the production of therapeutic proteins with fully human N-glycosylation profile and superior pharmacokinetic properties [69].…”

Innovative Metrics for Reporting and Comparing the Glycan Structural Profile in Biotherapeutics

“…Sialyl Lewis X is a member of blood carbohydrates present on the surface of different immune cells; it is a ligand for selectins and play an important role in different physiological phenomena [82,83]. Moreover, antennae fucosylation is also correlated with the rapid clearance and cellular uptake mediated by the mannose receptor present on macrophages and dendritic cells [8,84]. Antennae fucosylation can therefore increase the clearance and likely immunogenicity.…”