Structural and Physical Basis for Anti-IgE Therapy

Abstract

Omalizumab, an anti-IgE antibody, used to treat severe allergic asthma and chronic idiopathic urticaria, binds to IgE in blood or membrane-bound on B lymphocytes but not to IgE bound to its high (FcεRI) or low (CD23) affinity receptor. Mutagenesis studies indicate overlapping FcεRI and omalizumab-binding sites in the Cε3 domain, but crystallographic studies show FcεRI and CD23-binding sites that are far apart, so how can omalizumab block IgE from binding both receptors? We report a 2.42-Å omalizumab-Fab structure, a docked IgE-Fc/omalizumab-Fab structure consistent with available experimental data, and the free energy contributions of IgE residues to binding omalizumab, CD23, and FcεRI. These results provide a structural and physical basis as to why omalizumab cannot bind receptor-bound IgE and why omalizumab-bound IgE cannot bind to CD23/FcεRI. They reveal the key IgE residues and their roles in binding omalizumab, CD23, and FcεRI.

Figure 1. How IgE mediates an allergic reaction via interaction with its two receptors.

(Left) Interactions of membrane-bound IgE (mIgE, blue) with CD23 (tangerine) on B-cells regulates soluble IgE (sIgE) production. (Right) Cross-linking of IgE bound to FcεRI (scarlet) on mast cells or basophils by allergens (brown) triggers the release of mediators, causing allergy.

Figure 2. Conformational changes in the IgE-Fc upon binding its receptors.

Top: The Cε3 domains adopt a closed conformation when bound to CD23 (PDB 4gko), an open one when bound to FcεRI (PDB 1f6a), and a hybrid conformation with chain A “open” and chain B “closed” when IgE is free in solution (PDB 2wqr). Bottom: The Cε2 domains adopt a bent conformation with contacts to one of the Cε3-4 domains in free IgE (PDB 2wqr), becomes even more bent upon binding FcεRI (PDB 2y2q), but is extended when bound to two non-omalizumab anti-IgE molecules (PDB 4j4p).

Figure 3. The omalizumab-Fv region.

(a) Electrostatic potentials derived from the 2.42 Å crystal structure; arrows indicate the residues implicated in binding IgE from site-directed mutagenesis studies. (b) Packing and hydrogen-bonding interactions of the three histidines in the H3 loop. (c) Interactions of L1:Asp30 showing that its side chain points away from the protein surface. Omalizumab-Fv residues are in green and IgE residues in blue.

Figure 4. The IgE/omalizumab interface.

(a) Two omalizumab-Fab molecules (green) binding to two IgE Cε3-4 domains (marine/blue) with the Cε2 domains (cyan) in the extended conformation. (b) Omalizumab-binding site (yellow/wheat) on the IgE Cε3 domain.

Figure 5. Free energy contributions of IgE residues towards binding

(a) omalizumab-Fv, (b) CD23, and (c) FcεRI. Residues experimentally implicated in binding omalizumab are labeled.

Figure 6. Two omalizumab molecules bind to IgE in an extended Cε2 conformation with the Cε3-Cε4 dimer in a closed or open conformation.

(a) The Cε2 domains in the free IgE structure clashes with the second omalizumab-Fv domain after superposition of each Cε3 domain (blue) from the 1:2 IgE-Fc/omalizumab-Fv complex onto that from the free IgE structure (PDB 2wqr) and displaying only omalizumab-Fv. (b) Both omalizumab-Fv domains exhibit no clashes with IgE after one of the Cε3 domains from the 1:2 IgE-Fc/omalizumab-Fv complex is superimposed onto the closed Cε3 conformation of chain A in PDB 2wqr, whereas the other Cε3 domain is superimposed onto the open Cε3 conformation of chain B; only omalizumab-Fv and the Cε2 domains in the omalizumab–Fab/IgE-Fc complex are displayed, while the Cε2 domains in the free structure are hidden.

Figure 7. Why omalizumab-bound IgE cannot bind CD23 or FcεRI and why omalizumab cannot bind to receptor-bound IgE.

(a) CD23 (tangerine) clashes with omalizumab–Fab (green) after the IgE Cε3 domains from omalizumab-bound IgE are separately superimposed onto that from CD23-bound IgE (PDB 4gko), and displaying only the omalizumab-Fv. The IgE-Cε2 domains in the extended position are modeled by superimposing the IgE Cε3-4 domains from PDB 4j4p onto those in CD23-bound IgE and displaying only the IgE Cε2 domains. Omalizumab heavy atoms within 2.5 Å of heavy atoms in CD23 are depicted as spheres. (b) FcεRI (scarlet) clashes with the IgE-Cε2 domains (cyan) in the extended position after each IgE Cε3 domain from omalizumab-bound IgE is separately superimposed onto that from FcεRI-bound IgE (PDB 2y7q), and displaying only omalizumab-Fv. The IgE-Cε2 domains were modeled in an extended position by superimposing the IgE-Cε3-4 domains from PDB 4j4p over the FcεRI-bound IgE and displaying only the Cε2 domains in the 4j4p structure, while hiding the IgE-Cε2 domains from 2y7q. Omalizumab and IgE-Cε2 heavy atoms within 2.5 Å of heavy atoms from FcεRI are depicted as spheres. (c) FcεRI (scarlet) clashes with omalizumab–Fv (green) after each Cε3 domain from omalizumab-bound IgE is separately superimposed onto that from FcεRI-bound IgE (PDB 2y7q), and displaying only omalizumab-Fv. Omalizumab heavy atoms within 2.5 Å of heavy atoms from FcεRI or IgE-Cε2 are depicted as spheres. (d) Omalizumab–Fv (green) clashes with CD23 or the Cε2 domain after each Cε3 domain from omalizumab-bound IgE is separately superimposed onto that from the 1:1 IgE-Fc/CD23 complex (see text), and displaying only omalizumab-Fv. Omalizumab heavy atoms within 2.5 Å of heavy atoms from CD23 or IgE-Cε2 are depicted as spheres.