Omalizumab for pediatric asthma.

IMPORTANCE TO THE FIELD: Omalizumab is of proven efficacy in the treatment of severe allergic bronchial asthma and works through inhibiting the activity of IgE and the allergic immune mechanism IgE mediates. It has been demonstrated to be efficacious in children with asthma but is not approved by the FDA for use in children below 12 years of age. AREAS COVERED IN THIS REVIEW: Omalizumab is a 95% humanized monoclonal antibody that binds to circulating IgE at the same site on the Fc domain as the high-affinity IgE receptor, FcϵRI. This blocks the interaction between IgE and mast cells and basophils, thereby preventing the release of inflammatory mediators that cause allergic signs and symptoms. WHAT THE READER WILL GAIN: From the review of the literatures and statements from the FDA, Genentec and Novartis, the reader will gain a better appreciation of the value of omalizumab in treatment of severe asthma and the current status of its reported side effects. TAKE HOME MESSAGE: Omalizumab is of proven efficacy in adults and children with severe asthma and allows a markedly reduced dependence on oral and inhaled corticosteroids and decreased hospitalizations. A potential mechanism of omalizumab's effect on thrombus formation and cardiovascular effect is postulated.

MEP chromatography of antibody and Fc-fusion protein using aqueous arginine solution.

MEP HyperCel resin, one of the Protein-A mimetic columns, is designed to bind antibodies at physiological pH and elutes the bound antibodies at mildly acidic pH. We have tested aqueous arginine solution for washing and elution of the resin. To our surprise, bound antibody and Fc-fusion protein eluted at pH 7.0 using 1M arginine solution. Various solvent additives were then examined at pH 7.0. Among the tested additives, urea and arginine were the only additives that were effective in elution. Thus, urea and arginine at low concentrations were effectively used for washing the resin. NaCl and MgCl(2) at 0.1-1M and ethanol at 5-20% were not effective. Based on these observations, it appears that protein binds to MEP resin through both polar and hydrophobic interactions with some contribution of electrostatic interaction, which can be simultaneously reduced by arginine or urea. On the other hand, Mabsorbent, another Protein-A mimetic column, appears to be more non-specific and non-selective.

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells.

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.

[The Fc-dependent binding of gram-negative bacterial endotoxins by human blood polymorphonuclear leukocytes]. / Fc-zavisimoe sviazyvanie éndotoksinov gramotritsatel'nykh bakterii polimorfnoiadernymi leikotsitami krovi cheloveka.

The treatment of thin blood smears with antibodies to glycolipid of chemotype Re, conjugated with horseradish peroxidase, revealed that under physiological conditions about 3.5% of leukocytes bound endotoxin of gram-negative bacteria by means of the Fc-dependent mechanism. In addition, about 4.9% of leukocytes may bind endotoxin as the result of the treatment of blood smears with the preparation of glycolipid of chemotype Re. At the acute period of bacterial cerebrospinal meningitis leukocytes capable of binding endotoxin in the body or during the treatment of blood smears are practically absent. The conclusion was made that the binding of endotoxins by leukocytes had a protective character.

The enhancement of the human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells by trypsin treatment is ascribed to the binding of trypsin to the Fc fragment.

Human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells was remarkably enhanced by trypsin treatment of the cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of proteins of the cells treated with trypsin was the same as that of the cells treated with pronase, although pronase treatment had been shown to reduce the activity in our previous study (FEMS Microbiol. Lett. 123, 305-310, 1994). This contradiction was clarified by the finding that trypsin bound the Fc fragment more strongly than the cells, and a small amount of trypsin remained in the cells treated with trypsin and washed well. On the basis of these results, it was concluded that the enhancement of cell activity by trypsin treatment was ascribed to binding of the Fc fragment to trypsin remaining in the trypsin-treated cells.

Blockade of immunoregulatory Fc-signalling by HIV peptides: oligopeptides from HIV gp120 and gp41 bind the Fc portion of IgG and increase the in vitro anti-ssDNA response.

Concomitant ligation of antigen receptors with Fc-receptors negatively signals B cells. Antibodies to the Fc portion of IgG prevent this negative Fc-signalling, provided that these antibodies do not emit Fc signals. Prevention of Fc signals leads to augmented antibody responses to self and foreign antigens, and reduces the requirement for T cells by 10- to 100-fold in T cell-dependent antibody responses. In ELISA assays, peptides from conserved portions of the glycoproteins, HIV-1 gp120 or gp41 from HIV-1 and HIV-2 bind to the Fc portion of IgG, but do not bind the F(ab')2 portion of IgG. HIV-derived peptides, which bind to the Fc portion of IgG, augment the antibody-forming cell response to single-stranded (ss)DNA. The spontaneous response to ssDNA using spleen cells from young mice, and the response in the presence of exogenous DNA using spleen cells from old mice, are augmented to the greatest extent. These results demonstrate that HIV peptides bind to the Fc portion of IgG and augment immune responses to DNA; they suggest the possibility that blockade of the Fc portion of IgG antibodies is associated with a reduction in Fc-mediated regulation of anti-self responses. Blockade of regulatory Fc-signalling may account for increased circulating immunoglobulins and autoantibodies in clinical AIDS.

The specific blocking of an IgG dependent erythrophagocytosis assay by protein G and ELISA determination of in situ bound IgG on erythrocytes of normal donors.

We report the development of an in vitro erythrophagocytosis assay in which the level of phagocytosis reflects the number of IgG molecules bound to the erythrocyte. This assay is sensitive to 300 IgG per erythrocyte above background levels. Blocking the Fc of the bound immunoglobulin with protein G totally blocks macrophage recognition of the opsonized red cell and prevents Fc-gamma-dependent phagocytosis. An accurate, reliable, easily performed CELL-ELISA (cellular ELISA) for the determination of very low levels of IgG on the human erythrocyte membrane has been developed. This CELL-ELISA is based on the use of biotin conjugated to goat anti-human IgG (GaHIgG) and streptavidin conjugated to alkaline phosphatase. The CELL-ELISA can be accurately performed on either fresh or glutaraldehyde fixed erythrocytes. When a population of healthy young adults was studied an average of 126 +/- 14 IgG molecules per erythrocyte were detected.