Evidence for an Asialoglycoprotein Receptor on Nonparenchymal Cells forO-Linked Glycoproteins

Abstract

B cell-activating factor receptor 3 (BR3)-Fc is an IgG1-receptor dimeric fusion protein that has multiple O-linked glycosylation sites and sialylation levels that can vary in the manufacturing process. Increased sialic acid levels resulted from increased site occupancy with the O-linked N-acetylgalactosamine (GalNAc-Gal), but because the ratio of sialic acid per mole of oligosaccharide remained approximately 1, this led to increased asialo terminal GalNAc. Previous studies have demonstrated an effect of terminal asialo Gal or GalNAc on the clearance of glycoproteins due to uptake and degradation by lectin receptors in the liver. However, the previous studies examined N-linked oligosaccharides, and there are less data regarding O-linked oligosaccharides. The objective of these studies was to determine the effects on the pharmacokinetics and distribution of the asialo terminal GalNAc and varying amounts of sialic acid residues on BR3-Fc. The results of the data presented here suggest that exposed Gal on the desialylated BR3-Fc led to rapid clearance due to uptake and degradation in the liver that was associated with nonparenchymal cells. It is interesting to note that the data indicated a decreased clearance and increased exposure of BR3-Fc as the sialic acid levels increased, even though increased sialic acid was associated with increased asialo GalNAc. Therefore, the exposed GalNAc did not seem to play a role in the clearance of BR3-Fc; although the Gal linked to the hydroxyl group at position 3 may have prevented an interaction. Because we did not see uptake of desialylated BR3-Fc in hepatocytes where the asialoglycoprotein receptor is localized, this nonparenchymal cell lectin may have preference for O-linked glycoproteins.B cell-activating factor receptor 3 (BR3)-Fc is an IgG1-receptor dimeric fusion protein that is directed against B cell-activating factor (BAFF of the tumor necrosis factor ligand superfamily) (Gordon et al., 2003;Mackay and Ambrose, 2003;. BR3-BAFF interaction seems to be particularly important for the regulation of B cell survival and maturation in the spleen (Ng et al., 2004). Prevention of BAFF binding to BR3 on B cells by neutralizing BAFF via BR3-Fc results in notable peripheral and tissue B cell reduction in rodents and monkeys due to apoptosis (Vugmeyster et al., 2006;Lin et al., 2007). BR3-Fc consists of two polypeptide chains, each containing 299 amino acids, with sequences from the extracellular domain of the human BAFF receptor BR3 and the Fc domain of human IgG1. The two chains are joined by two disulfide bonds located in the IgG1 Fc domain near the junction between the BR3 extracellular domain and the IgG1 Fc region.In addition, BR3-Fc has multiple O-linked glycosylation sites with sialylation on the glycoproteins that can vary in the manufacturing process. There are six potential sites per BR3 domain and two BR3 domains per molecule. The monosaccharide analyses of BR3-Fc lots indicate that, on average, less than half of the identified O-linked sites are occupied and th...

Biodistribution of [125I]-labeled therapeutic proteins: Application in protein drug development beyond oncology**All authors are current or former employees of Wyeth, Inc.

“…The list of published studies on biodistribution of therapeutic proteins is gradually increasing but is still relatively sparse, with majority of studies focusing on tumor‐targeting agents. Several studies on biodistribution of therapeutic proteins beyond oncology have been published in the recent years to address a variety of issues, such as FcRn‐mediated effects,10 carbohydrate‐related effects,11, 12 and penetration across the blood–brain barrier 13…”

Sialidase Unmasks Mucin Domain Epitopes of Lubricin

“…23,[26][27][28][29][30][31][32][33] Thus, sialylation masks the terminal galactose residues, thereby protecting glycoproteins from hepatocyte and non-parenchymal liver cell internalization and subsequent lysosomal degradation. 24,34 Also, lubricin derived from osteoarthritis synovial fluid contains more monosialylated glycans compared with disialylated glycans in rheumatoid arthritis (RA) synovial fluid. 13 Given our data in which the S6.79 epitopes on lubricin molecules in normal plasma and serum are more sialylated than those in normal synovial fluid, it is possible that the increased sialylation in RA synovial fluid could be due to an increased presence of blood-derived lubricin.…”

Antibody Glycosylation and Its Impact on the Pharmacokinetics and Pharmacodynamics of Monoclonal Antibodies and Fc-Fusion Proteins

“…There are several other Fc‐fusion molecules that also rely on sialylation in reducing the in vivo clearance. BR3–Fc‐fusion has multiple O‐linked glycosylation sites, and it has been shown that changes of the sialic acid content through the manufacturing process have a direct relationship with the PK properties . BR3–Fc molecules with high sialic acid content exhibited much higher systemic exposure following i.v.…”

“…Some therapeutic mAbs also bear additional glycosylation in the Fab domain such as cetuximab at Asn 88 of the VH region . In addition, some of the Fc‐fusion partner molecules such as etanercept and B cell‐activating factor receptor 3 (BR3)–Fc also possess O‐linked glycans . Alteration of glycan compositions and structures can cause conformational changes of the Fc domain, which could change binding affinity to Fcγ receptors, resulting in changes of immune effector functions .…”