Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).

No abstract

The erythropoietin (EPO) molecule contains four carbohydrate chains. Three contain N-linkages to asparagines at positions 24, 38, and 83, and one contains an O-linkage to a serine at position 126. We constructed human EPO variants that eliminated the three N-glycosylation sites by replacing the asparagines with glutamines singly or in combination. The O-linked carbohydrate chain was removed by replacing the serine with glutamine, valine, histidine, or alanine. A variant with a double mutation (Gln38,83) and another with a triple mutation (Gln24,38,83) were secreted poorly from COS1 and CHO cells even though RNA encoding these variants was present. All other variants with mutations in N-linked glycosylation sites were secreted normally. Removal of any of the N-glycosylation sites reduced the in vivo but not the in vitro biological activity of the EPO molecule. All the mutations at Ser126, the O-glycosylation site, were secreted normally. In vitro activity was also unaffected except for Ala126 which had a 50-fold decrease. The Val126 variant was tested in vivo, and its specific activity was only slightly less than that of the native EPO, which indicates that the O-linked carbohydrate is not essential for activity.

Oligomerization of cytokine receptors including the erythropoietin (EPO) receptor has been advanced as a model for activation. If homodimerization of the EPO receptor activates it, then bivalent antibodies raised to the extracellular domain of the EPO receptor should also homodimerize and activate. Mouse monoclonal antibodies (IgG) raised to the soluble, extracellular domain of the human EPO receptor (EPOR) were found that would stimulate thymidine uptake of an human EPO-dependent cell line, UT-7/EPO. Dose response curves showed bell shapes where activity was low at low and high concentrations. Monovalent (Fab) fragments bound to the receptor but did not stimulate thymidine uptake, which indicates that two antibody binding sites are required for activation. The anti-EPOR antibodies stimulated the formation of burst forming unit erythroid colonies from human CD34 ؉ cells purified from peripheral blood. This indicates that homodimerization of the EPO receptor by anti-EPOR antibodies is sufficient for both proliferation and differentiation of erythroid progenitor cells and that the constraints on dimerization necessary for activation are rather loose. Erythropoietin (EPO)1 is a glycoprotein hormone that is the primary regulator of erythropoiesis. It stimulates erythroid progenitors to proliferate and differentiate via binding to and activation of an EPO receptor expressed on the surface of cells. The murine and human EPO and EPO receptor genes have been cloned (1-6). The human EPOR gene encodes a 508-amino acid protein that includes a 25-amino acid signal peptide, a 225-amino acid extracellular domain, a 22-amino acid transmembrane domain, and a 236-amino acid cytoplasmic domain. The EPO receptor is a member of a family of cytokine receptors that includes receptors for prolactin, growth hormone, interleukins 2-7, granulocyte macrophage colony-stimulating factor, granulocyte colony-stimulating factor, leukemia inhibitory factor, thrompopoietin ligand, and ciliary neurotrophic factor (7-11). This family is characterized by regions of similarity in their extracellular and intracellular domains. The family is also characterized by a lack of an identifiable protein tyrosine kinase domain in their intracellular region. Activation of the receptors by ligand binding induces a cascade of signaling events including phosphorylation of the EPO receptor (12), activation of the JAK-STAT pathway (13,14), activation of PI3 kinase (15,16), and activation of the RAS-MAPK pathway (17). Down modulation of the signal transduction pathway is also effected by binding of the protein phosphatase SH-PTP-1 to the C-terminal region (18).Activation of many different receptors is thought to occur by oligomerization of the receptors (for reviews see Refs. 19 and 20). One of the most studied systems is growth hormone receptor where complexes have been shown to consist of two receptors bound to one ligand (21,22). In this case the ligand is thought to act as a cross-linker bringing the receptors into close proximity whereupon they bind and intera...

N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn 123 -Thr 125 ) introduced into a position close to the existing O-glycosylation site (Ser 126 ) had an additional O-linked carbohydrate chain and not an additional Nlinked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.Secreted proteins are often glycosylated during transit through the secretory apparatus in eukaryotic cells. These carbohydrates can be attached to the hydroxyl group on a serine or threonine (O-linked glycosylation) or the amine of an asparagine via an N-glycosidic bond (N-linked glycosylation). The addition of carbohydrate chains to the polypeptide backbone of a protein may have an impact on the structure, solubility, antigenicity, folding, secretion, and stability of the protein (1-8). The carbohydrate may also affect the clearance rate and in vivo activity of the protein (9 -12).The nature of the signal for carbohydrate addition is partially understood. N-Linked carbohydrate addition is mediated by oligosaccharide transferase and occurs at asparagine residues that are part of the consensus sequence Asn-Xaa-(Ser/ Thr), where Xaa can be any amino acid, except proline (13-16). The observation that not all consensus sequences are glycosylated suggests that there are additional sequence or conformational requirements essential for efficient carbohydrate attachment (17,18). Although at least 12-14 amino acids must be synthesized and have entered the luminal surface of the endoplasmic reticulum for carbohydrate addition, the synthesis of the protein need not be completed for glycosylation to take place (19,20). This suggests that the structures for carbohydrate addition are recognized in partially folded molecules. The sequence context of the glycosylation site has also been shown to influence the efficiency of glycos...