Project Summary/Abstract Antibodies are an important class of glycoproteins. Compelling experimental data have shown that the fine structures of the highly conserved Fc glycans in IgG type antibodies can have profound effects on the antibody’s biological functions and, in the case of therapeutic antibodies, the clinical efficacy, such as antibody-dependent cellular cytotoxicity (ADCC), complement- dependent cytotoxicity (CDC), activation of apoptosis, and anti-inflammatory activities. More recently, it was reported that a single oligomannose type glycan at the IgE-Fc domain was responsible for high-affinity binding to IgE receptor on mast cells and was critical for initiation of allergic reactions. However, progress in understanding the functional roles of antibody glycosylation is hampered by the tremendous structural heterogeneity of Fc glycosylation. Gnetic approach to glycoengineering has achieved some successes but the antibody glycoforms that can be generated by genetic approach are limited. Currently there is no general method avaiable to control glycosylation to a pre-defined homogeneous form during recombinant antibody production. In this application, we propose to explore a chemoenzymatic method that we recently developed to make various homogeneous IgG and IgE glycoforms. Through performing Fc receptor binding studies, we aim to understand how different glycan structures modulate the effector functions of IgG and IgE and to apply the knowledge obtained for developng better therapeutics. Three specific aims are proposed to achieve the goal. Aim 1 is to further explore the chemoenzymatic method for antibody glycoengineering by investigating enzyme structures, searching for new enzymes and mutants, and evaluating new glycan substrates. Aim 2 is to establish a general site-specific conjugation method for constructing homogeneous antibody-drug conjugates. Functional tags will be introduced to Fc glycan site- specifically and a toxin will be chemoselectively ligated to the Fc glycan to provide homogeneous ADCs. Aim 3 is to perform site-specific glycosylation remodeling of IgE Fc for probing the glycosylation functions in allergic reactions. A successful pursuit of the proposed research could lead to the creation of new IgG and IgE glycoforms with novel biological properties. The chemoenzymatic method may become a general platform technology for site- specific antibody-drug conjugation and labeling. The study on IgE Fc glycosylation, if successful, will fill a gap of our knowledge in glyco-immunology related to allergic reactions and diseases.