Abstract
Immunoglobulin E, one of the five classes of antibodies, plays a central role in mediating allergen-induced allergic responses, leading to the sensitization of mast cells and basophils, their discharge of a host of pharmacological mediators, and the manifestation of various allergic symptoms. Active pharmaceutical programs have focused on developing drugs that neutralize or block the mediators released from those two types of inflammatory cells. However, such drugs are often not effective in treating severe allergic diseases. In 1987, Dr. Tse-Wen Chang, who is my thesis supervisor, invented an anti-IgE antibody with a unique set of binding specificities: it binds to free IgE in blood and extravascular space and to membrane-bound IgE on B cells, but not to IgE bound by Fc□RI on mast cells and basophils. One of such antibodies, Omalizumab, has been studied in more than 30 phase II and III clinical trials in various allergic indications and proven to be efficacious and safe in treating allergic asthma, allergic rhinitis, and other allergic diseases. Omalizumab has been approved in the U. S. A., EU, and some other countries for treating moderate-to-severe asthma. It was also approved in Taiwan in past April. Since the most important property of the anti-IgE therapeutic is its unique binding specificity, it is of great interest to understand the structural aspects in the interaction between anti-IgE and IgE. It is now clear that Omalizumab binds to human IgE on the CH3 domain. Previous molecular simulation and molecular docking studies indicated that the anti-IgE binding site on IgE is adjacent to or overlaps with the Fc□RΙ binding site on CH3. As an IgE molecule is bound by Fc□RI, the binding site for Omalizumab is hindered or masked and can not be bound by Omalizumab. Thus, Omalizumab cannot cross-link IgE bound on Fc□RI and sensitize basophils and mast cells. While substantial progress has been made in delineating and dissecting the interaction between IgE and Fc□RI, limited knowledge has been obtained regarding the interaction between IgE and anti-IgE. Fc□RI and IgE bind to each other in 1:1 stoichiometry: one Fc□RI molecule can bind to both CH3 domains simultaneously in an asymmetrical manner. In contrast, two Omalizumab molecules can bind to one IgE, and one Omalizumab can bind to two IgE molecules. It has been found that anti-IgE and IgE form 2:1, 1:2, or 3:3 immune complexes in the blood. These results indicate that each □ chain of IgE can distinctively bind to one anti-IgE molecule. To further analyze the interaction between IgE and Omalizumab, we have expressed various monomeric and dimeric fragments of IgE.Fc and studied their interaction with Omalizumab. The ELISA results indicated that a single CH3 domain on one □ chain can bind to Omalizumab. We also used SPR technique to measure and compare the binding affinities of these IgE.Fc fragments with Omalizumab and their respective binding kinetics. The SPR analysis indicated that Omalizumab binds to a single CH3 domain with affinity comparable to that to intact IgE. The sensorgram also showed that one dimeric IgE molecule can bind to two immobilized Omalizumab molecules while a monomeric CH3 domain can bind to one. These results demonstrated that IgE can bind to Omalizumab through a single CH3 domain with comparable affinity as intact IgE. Furthermore, the results also suggest that the two binding sites on IgE for Omalizumab are independent and that the contact residues of each site should be completely located on one CH3 domain.