Theses and Dissertations from UMD

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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM

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Author: search.filters.author.Ou, Chong

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    CHEMOENZYMATIC FC GLYCAN ENGINEERING FOR IMPROVING ANTIBODY IMMUNOTHERAPY
    (2022) Ou, Chong; Wang, Lai-Xi; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    IgG antibodies contain a conserved N-glycan on the Fc domain. The structures of the glycan play an important role in modulating an antibody’s effector functions. The Fc N-glycans also provide a suitable site for functionalization and conjugation of antibodies in a site-specific manner. The Wang lab have recently developed a general chemoenzymatic method for Fc glycan remodeling through endoglycosidase-based deglycosylation and reglycosylation. My thesis research focuses on three projects: Project 1, development of a site-selective conjugation method for synthesizing antibody-drug conjugates (ADCs); Project 2, application of the method for improving antibody’s complement-dependent cytotoxicity (CDC); Project 3, exploration of a dual functionalization method for enhancing internalization and lysosomal delivery of antibodies.Optimizing the synthesis for site-specific antibody conjugates using the glycan remodeling strategy is the first part of my thesis. We developed a facile synthetic strategy to functionalize glycan oxazolines from sialoglycan, which are the key donor substrates for enzymatic Fc glycan remodeling. An efficient chemoenzymatic method based on the EndoS2-D184M was also developed to functionalize therapeutical antibodies with different Clickable groups including azide-, cyclopropene-, and norbornene-tags. Homogenous antibody-drug conjugates (ADCs), with drug-antibody ratio of 4 were successfully obtained through three different Click reactions on the tags introduced. Comparison experiments indicated that the ADCs generated by these three Click reactions showed potent cancer cell killing activity and excellent serum stability. Complement-dependent cytotoxicity (CDC) is a major effector function for antibodies to deplete target cells. But for the IgG antibodies, which is the most widely used isotype for therapeutic antibodies, potent complement activation is restricted. With our optimized conjugation method, we constructed structurally well-defined antibody-αGal and antibody-rhamnose conjugates, which were designed to recruit natural anti-αGal and anti-rhamnose antibodies for enhancing CDC, using trastuzumab as a model antibody. Our preliminary in vitro study indicated that the antibody-rhamnose cluster conjugates could mediate potent CDC activity against targeted cancer cell with high selectivity. Since the rate of receptor internalization is a key factor for the selection of druggable antigen, enhancing the internalization efficiency could improve the efficacy of the ADC and possibly broaden the druggable antigens for ADCs. At the same time, the lysosomal delivery of ADCs could enhance their pharmaceutical efficacy. Therefore, we introduced a pair of orthogonal Click groups on the sialo-complex type glycan (SCT), and we used one of the clickable groups to ligate the drug, while using another one to carry an internalizing factor. This platform provided great flexibility to test out different combinations of antibodies, cytotoxic drugs, and internalizing factors. To date, preliminary cell-based studies have indicated that could improve the toxicity of a cetuximab based ADC with mannose-6-phosphate as an auxiliary internalization factor.