Design And Preparation Of Anti-IgE Antibody

It is well known that IgE and its high affinity receptor FcεRI are key molecules in triggering allergic diseases. Novel anti-allergy drugs targeting IgE have developed quickly, in which humanized anti-IgE monoclonal antibody Omalizumab (Xolair?) has been authorized by FDA for clinical treatment and showed prominent curative effect in adult moderate or severe asthma. By far, there was no similar research in China,and so, it was significant to obtain novel anti-IgE antibodies.A series of researches have showed that functional anti-IgE antibodies could produce marked effect against allergy diseases by blocking the formation of IgE/FcεRI complex. With the development of NMR and X-ray diffraction, the crystal structure of IgE-Fc and FcεRI were resolve precisely, which offered significant information for designing new drugs. In this thesis, based on the crystal structures of IgE-Fc, FcεRI and IgE-Fc/FcεRI complex, the functional epitope of IgE to bind FcεRI and the functional anti-IgE antibody MAE11 was analyzed theoretically. According to the predicted results, two IgE-Fc mutants, fragments E24 (IgE Cε2-Cε4) and E34 (IgE Cε3-Cε4), were cloned, expressed and identified. The experimental results were consistent with the theoretical conclusion. Furthermore, the extracellular part of FcεRIαwas synthesized and subsequently membrane FcεRIα-bearing cell model was established for screening and identifying new anti-IgE antibodies. Using the constructed model, six anti-IgE mouse antibodies were obtained and evaluated. In detail, the research results were shown as follows:1. To analyze the functional epitope of IgE theoretically: Based on the crystal structure of IgE-Fc/FcεRI complex, the conformations of the monomer E24, E34 and FcεRI were constructed using computer aided homology modeling and optimized with molecular mechanism method. The chosen methods were evaluated and the theoretical structures were determined. Under the same force field and methods, the 3-D theoretical structure of MAE11, the mouse monoclonal antibody (MAb) of Omalizumab, was obtained. Dealing with the distance geometry, intermolecular hydrogen bond forming and hydrophobic-hydrophilic interaction methods, the functional epitopes and interaction mode of E24/FcεRI, E34/FcεRI, E24/MAE11 and E34/MAE11 complexes were predicted. The functional epitope of IgE was considered in IgE Cε3 domain.2. To confirm the functional epitope of IgE experimentally: IgE fragments E24 and E34 were cloned and expressed using molecular biological technology. E24 and E34 could bind FcεRI and Omalizumab, indicating that the functional epitope of IgE was included in IgE Cε3-Cε4 domain, which was consistent with the theoretical results.3. Establishment and identification of cell model for screening functional anti-IgE antibody: By analyzing the orientation tendency of different transmembrane domains, we concluded the rule of membrane spanning mode, with which the transmembrane domain of Her2 was selected to achieve the surface display of FcεRIα. And then, the stable membrane FcεRIα-expressing cell line was established and verified with Omalizumab which could antagonize with FcεRI to bind soluble IgE but did not bind membrane FcεRI-loaded IgE.4. Preparation and preliminary evaluation of mouse anti-IgE MAbs: Mice were immunized with E34, and after cell fusion and clone screening, six mouse anti-human IgE MAbs were obtained (named as QME1~6), in which only QME5 could bind membrane IgE. In addition, the epitopes of five MAbs except QME3 were overlapped with Omalizumab. QME1~6 could not bind membrane FcεRI-loaded IgE, indicating that none of them had potential hypersusceptibility. Meanwhile, both QME5 and QME6 could block the formation of IgE/FcεRI complex. However, due to the low affinity to bind IgE (the affinity constant was 1×107 M-1), QME5 could antagonize FcεRI only in a high molar ratio to IgE.In conclusion, with the computer aided homology modeling techniques and theoretical analysis methods, the potential functional epitope and interaction mode between receptor and ligand, antibody and antigen could be determined theoretically. Furthermore, the results mentioned above offered salutary highlights to rationally design novel antagonists including functional antibodies targeting to IgE.