Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc␥RI, Fc␥RIIA, Fc␥RIIB, Fc␥RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc␥R; Fc␥RII and Fc␥RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc␥RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc␥RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.

Immunoglobulins (IgG) are soluble serum glycoproteins in which the oligosaccharides play significant roles in the bioactivity and pharmacokinetics. Recombinant immuno-globulins (rIgG) produced in different host cells by recombinant DNA technology are becoming major therapeutic agents to treat life threatening diseases such as cancer. Since glycosylation is cell type specific, rIgGs produced in different host cells contain different patterns of oligosaccharides which could affect the biological functions. In order to determine the extent of this variation N-linked oligosaccharide structures present in the IgGs of different animal species were characterized. IgGs of human, rhesus, dog, cow, guinea pig, sheep, goat, horse, rat, mouse, rabbit, cat, and chicken were treated with peptide-N-glycosidase-F (PNGase F) and the oligosaccharides analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for neutral and acidic oligosaccharides, in positive and negative ion modes, respectively. The data show that for neutral oligosaccharides, the proportions of terminal Gal, core Fuc and/or bisecting GlcNAc containing oligosaccharides vary from species to species; for sialylated oligosaccharides in the negative mode MALDI-TOF-MS show that human and chicken IgG contain oligosaccharides with N-acetylneuraminic acid (NANA), whereas rhesus, cow, sheep, goat, horse, and mouse IgGs contain oligosaccharides with N-glycolylneuraminic acid (NGNA). In contrast, IgGs from dog, guinea pig, rat, and rabbit contain both NANA and NGNA. Further, the PNGase F released oligosaccharides were derivatized with 9-aminopyrene 1,4,6-trisulfonic acid (APTS) and analyzed by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-LIF results indicate that the proportion of the two isomers of monogalactosylated, biantennary, complex oligosaccharides vary significantly, suggesting that the branch specificity of beta1, 4-galactosyltransferase might be different in different species. These results show that the glycosylation of IgGs is species-specific, and reveal the necessity for appropriate cell line selection to express rIgGs for human therapy. The results of this study are useful for people working in the transgenic area.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

Therapeutic glycoproteins produced in different host cells by recombinant DNA technology often contain terminal GlcNAc and Gal residues. Such glycoproteins clear rapidly from the serum as a consequence of binding to the mannose receptor and/or the asialoglycoprotein receptor in the liver. To increase the serum half-life of these glycoproteins, we carried out in vitro glycosylation experiments using TNFR-IgG, an immunoadhesin molecule, as a model therapeutic glycoprotein. TNFR-IgG is a disulfide-linked dimer of a polypeptide composed of the extracellular portion of the human type 1 (p55) tumor necrosis factor receptor (TNFR) fused to the hinge and Fc regions of the human IgG(1) heavy chain. This bivalent antibody-like molecule contains four N-glycosylation sites per polypeptide, three in the receptor portion and one in the Fc. The heterogeneous N-linked oligosaccharides of TNFR-IgG contain sialic acid (Sia), Gal, and GlcNAc as terminal sugar residues. To increase the level of terminal sialylation, we regalactosylated and/or resialylated TNFR-IgG using beta-1,4-galactosyltransferase (beta1,4GT) and/or alpha-2,3-sialyltransferase (alpha2,3ST). Treatment of TNFR-IgG with beta1,4GT and UDP-Gal, in the presence of MnCl(2), followed by MALDI-TOF-MS analysis of PNGase F-released N-glycans showed that the number of oligosaccharides with terminal GlcNAc residues was significantly decreased with a concomitant increase in the number of terminal Gal residues. Similar treatment of TNFR-IgG with alpha2,3ST and CMP-sialic acid (CMP-Sia), in the presence of MnCl(2), produced a molecule with an approximately 11% increase in the level of terminal sialylation but still contained oligosaccharides with terminal GlcNAc residues. When TNFR-IgG was treated with a combination of beta1,4GT and alpha2,3ST (either in a single step or in a stepwise fashion), the level of terminal sialylation was increased by approximately 20-23%. These results suggest that in vitro galactosylation and sialylation of therapeutic glycoproteins with terminal GlcNAc and Gal residues can be achieved in a single step, and the results are similar to those for the stepwise reaction. This type of in vitro glycosylation is applicable to other glycoproteins containing terminal GlcNAc and Gal residues and could prove to be useful in increasing the serum half-life of therapeutic glycoproteins.

No abstract

The selectins are cell adhesion molecules whose carbohydrate-binding domain (C-type lectin) is thought to be involved in leukocyte adhesion to activated vascular endothelium in the inflammatory process. A series of peptides, based on a conserved region (48YYWIGIRK55-NH2) of the lectin domain of E-, L- and P-selectins, were analysed for their ability to block selectin-mediated cell adhesion in vitro, and neutrophil infiltration into sites of inflammation in vivo. The peptides inhibited the adhesion of myeloid cells to recombinant forms of E- and P-selectin. The adhesion of myeloid cells to human endothelial cells, stimulated to express E-selectin, was also inhibited by the peptides. Finally, the peptides blocked the adhesion of lymphocytes, expressing L-selectin, to high endothelial venules in lymph nodes which contain the ligand for L-selectin. A clear structure-activity relationship was established when peptides of different amino acid chain lengths were tested in these assays. Peptides lacking tyrosine residues (e.g. WIGIR-NH2) at their amino terminus were poor inhibitors of selectin-mediated cell adhesion in vitro. The peptides that were found to be inhibitors of cell adhesion in vitro were also found to inhibit (up to 70%) neutrophil infiltration into sites of inflammation in a thioglycollate-induced peritonitis mouse model system. They also significantly reduced (> 50%) the migration of neutrophils into cytokine-treated skin. These results strongly suggest that compounds based on these tyrosine-containing, selectin-derived peptides could be used as anti-inflammatory therapeutic agents.

No abstract

Previously, it was established that the peptide YYWIGIRK-NH2 inhibits both myeloid cell adhesion to selectins in vitro and neutrophil influx into inflammatory sites in vivo (Briggs et al., 1995). Initial structure/activity studies revealed that at least one Y residue at the N-terminus of the peptide was essential for these bioactivities but that the C-terminal K residue was unnecessary for inhibitory activity. We have now synthesized a new series of peptides which contain single residue substitutions at each position of the reference peptide, YYWIGIR-NH2, and have tested these peptides for inhibitory activity in a selectin cell binding assay. In addition, peptides containing single D-amino acids at selected positions, or an all D-configured reference peptide sequence, or the retro-inverso version (rigiwyy-NH2) of the reference peptide sequence have also been analyzed for inhibitory activity in the same assays. Finally, the ability of the reference peptide and a specifically designed control sequence (YY(AIB)IGIR-NH2) to discriminate between potential synthetic saccharide ligands, including sialyl-Lewis x, Lewis x, and sialyl-N-acetyl-lactosamine, was investigated using isothermal titration calorimetry. The results of these studies demonstrate that whereas many single amino acid substitutions are tolerated in the peptide without complete loss of inhibitory activity, substitution at some positions (e.g., the W residue) results in relatively inactive compounds, clearly pointing to the importance of these residues in making critical contacts with the appropriate saccharide ligand. Titration calorimetry revealed that the reference peptide does not discriminate between Lewis x or sialyl-Lewis x in vitro, but binds these saccharides with nearly 40-fold higher affinity (KD 25 microM) than the nonfucosylated trisaccharide, sialyl-N-acetyl-lactosamine. We can infer from these studies that the presence of a sialyl group per se, is not a requisite for complex formation between the reference peptide and its saccharide ligand. Substitution of single D-amino acid residues at various positions in the reference peptide sequence reduces or eliminates all inhibitory properties. However, the all D-configured peptide or the retro-inverso peptide sequence have greater activity than the all L-configured reference peptide in the in vitro biological assays, and each was an effective inhibitor of neutrophil infiltration in a thioglycolate-induced mouse peritonitis model. These results, combined with the results of titration, allow us to conclude that binding between the reference peptide and its saccharide ligand, which affords its inhibitory properties, is mediated by the presence of a contiguous, nonpolar surface, or face, presented at the N-terminus of the reference peptide, likely encompassing the sequence YYWI. Furthermore, the W plays a critical role in binding, probably through formation of an essential hydrogen bond with a suitably juxtaposed group carried on the saccharide ligand.