Characterization of Complex Formation by Humanized Anti-IgE Monoclonal Antibody and Monoclonal Human IgE

Abstract

The interaction of human IgE with high-affinity IgE Fc receptors on cells of the immune system plays an essential role in the type I hypersensitivity reaction. A proposed therapy is to use an anti-IgE monoclonal antibody to block the binding of IgE to its high-affinity receptor on mast cells and basophils, thus preventing subsequent release of the inflammatory agents after exposure to allergen. We report here the solution characteristics of immune complexes formed by a humanized anti-IgE monoclonal antibody (rhuMAb E25) and IgE using sedimentation analysis and size exclusion chromatography. We demonstrate that the rhuMAb E25 is able to form a variety of complexes with IgE at different molar ratios. The largest complex was identified by sedimentation equilibrium analysis as a heterohexamer with very high stability. The intermediate complex formed when one of the interacting components is in large molar excess appears to have a trimeric structure. The high-affinity interaction of rhuMAb E25 and IgE has also been confirmed. Furthermore, by using hydrodynamic modeling, we show that the largest complex may be represented by a cyclic structure.

Effect of antigen binding affinity and effector function on the pharmacokinetics and pharmacodynamics of anti-IgE monoclonal antibodies

“…Liu et al demonstrated that omalizumab and IgE formed complexes of various sizes in vitro due to two potential omalizumab binding sites on IgE. 7 The size of the anti-IgE:IgE complexes formed were dependent on the molar ratio of the interacting components, with hexamers formed when omalizumab was in equal molar ratio with IgE and trimers observed when omalizumab was in 5-fold molar excess to IgE. Complex size distribution was also evaluated for both HAE1 and HAE2 prior to conducting our in vivo studies.…”

“…6 Data from studies in mice, monkeys and humans have shown that the disposition of omalizumab is influenced by both its IgG1 framework and by complex formation with IgE. [7][8][9] The substantial structural homology between HAE1 and omalizumab and the similarities in their mechanism of action suggest that the absorption, distribution, metabolism, and excretion (ADME) profiles should be similar. The markedly higher affinity of HAE1 for IgE, however, may modify the pharmacodynamic (PD) profile of both free IgE and total IgE (free IgE and IgE complexed with drug) compared with omalizumab.…”

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

“…It is likely that the affinity of an anti-IgE molecule towards IgE was higher in serum compared to that in PBS and that the largest anti-IgE:IgE complex observed in serum was smaller than expected. This higher observed affinity is probably due to the crowding of macromolecular components in serum as discussed by Minton et al (Minton 2005;Zhou et al 2008 (Liu et al 1995). b Differential sedimentation coefficient distribution of IgE (solid line) and anti-IgE (dotted line) monomers at 0.64 mg/mL (I); IgE and anti-IgE complexes at various molar ratios (II and III) in PBS at 10°C.…”

“…Since the antiIgE MAb has two antigen binding sites each of which could combine with one of two sites on the target IgE molecule (located on each Fc heavy chain), the complexes could become very large. SV-AUC and SE-AUC measurements were used to determine weight-average molecular weight and size distribution to clearly show that this did not happen, and that the complexes that formed were of limited size (Liu et al 1995). The formation of these complexes, which are dependent on the molar ratio of IgE:anti-IgE (Fig.…”

“…3:l (dash-dotted line), 6:1 (dashed line), and 10:1 (dotted line). The sedimentation coefficients have been corrected to the standard condition of water at 20 C. No faster moving species was observed in early scanning (previously published in Liu et al 1995) conditions. Direct characterization in serum may now allow for better drug candidate selection and should be carefully considered during molecular assessment in early research stages.…”