Study On The Structure And Function Of Novel Anti-IgE Humanized Monoclonal Antibody

Object: IgE is the central macromolecular mediator responsible for the progression of allergic reactions. Omalizumab (Xolair) is a humanized monoclonal anti-IgE antibody directed at the FcεRI binding domain of human IgE, which represents a novel therapeutic approach in the management of asthma.Methods: We developed monoclonal antibody against human IgE via hybridoma technique. In an attempt to minimize the immunogenicity of murine monoclonal antibody for potential clinical application, a humanized antibody was constructed by complementary determining region (CDR) grafting. The function of humanized antibody was determined through ELISA assay, flow cytometry analysis and cell degranulation assay in vitro. Bio-panning of PDPLs with anti-IgE antibody was performed to select peptides binding to Hu7A5m18. The selected epitope was analysed based on the crystal structure of IgE-FcεRIαcomplex.Results: In this study, we developed a monoclonal antibody (7A5) against human IgE via hybridoma technique. In an attempt to minimize the immunogenicity of murine 7A5 (m7A5) for potential clinical application, a humanized version of 7A5, denoted as Hu7A5m18, was successfully constructed by complementary determining region (CDR) grafting. Our data showed that Hu7A5m18 could inhibit free IgE molecules to bind to receptors without affecting IgE already bound to cellular receptors. Importantly, Hu7A5m18 was able to inhibit IgE-induced histamine release of basophilic leukemia cells. The affinity of m7A5, Hu7A5m18 and Omalizumab were analysed using a BIAcore T100 analyser. The KD were 1.9nM, 1.7nM, 23.8nM respectively.Next, the phage display peptide library technology was employed to select peptides binding to Hu7A5m18 and a striking peptide sequence motif was recovered, which is homologous to the sequence 391KQR393 within the Cε3 domain of IgE-Fc, Our results further indicated that Hu7A5m18 specifically bound to the synthesized peptide"388KEEKQRN394"containing the 391KQR393 motif in IgE-Fc. The epitope of Hu7A5m18 was found to be spatially close to the FcεRI-binding site, suggesting that Hu7A5m18 binding to IgE might block IgE binding to receptors via steric hindrance. In our previous study, the Omalizumab epitope has been indentified to be the 424HLP426 motif within the Cε3 domain of IgE-Fc, which overlaps with the high-affinity IgE receptor-binding site. We compared the binding site of FcεRIα-chain to IgE-Fc with the localization of the Hu7A5m18 epitope identified in this study. The Hu7A5m18 epitope is spatially close to the FcεRI-binding site, which explains why Hu7A5m18 can inhibit free IgE molecules to bind to receptors without affecting IgE already bound to cellular receptors. Moreover, because Hu7A5m18 and Omalizumab epitope are also spatially close, Hu7A5m18 could compete with Omalizumab in binding to human IgE.Conclusion: The present study shows that a new anti-IgE humanized anibody, Hu7A5m18, tightly binds to free circulating IgE molecules and prevents binding of these molecules to receptors. Our results provide first evidence that in IgE-sensitized cells the mAb Hu7A5m18 efficiently and dose-dependently inhibits anti-IgE-triggered degranulation. The 391KQR393 motif within the Cε3 domain of IgE-Fc was identified as the 7A5 epitope in the present study and found to be spatially close to the FcεRI-binding site, suggesting that 7A5 binding to IgE might block IgE binding to receptors via steric hindrance. Taken together, the anti-IgE monoclonal antibody 7A5 may have the potential to be developed as a therapeutic agent for the treatment of allergic diseases.