Exploring functionalized sugar nucleotides for cell surface engineering and construction of antibody-drug conjugates (ADCS)
MetadataShow full item record
Glycans mediate a wide range of cellular functions, such as cell-cell interactions, adhesion, and differentiation, by mediating intracellular signaling events. In addition, glycosylation plays an important role inside cells by regulating protein trafficking, turnover and signal transduction. Specifically, sialic acids are essential in mediating a variety of physiological and pathological processes, whose generation is governed by the sialic acid biosynthetic machinery. This dissertation has taken advantage of different functionalized CMP-sialic acids, combined with various click reactions, to achieve efficient cell surface glycoprotein labeling and construction of Antibody-Drug Conjugates (ADCs). We have developed SEEL (selective exoenzymatic labeling) methodology to install chemical reporters on cell surface acceptor glycans using azide modified CMP-sialic acid, which only labels a specific class of cell surface molecules (e.g., N- vs O-glycans). In light of the direct nature of this method, we further developed a more efficient strategy, termed one-step SEEL, by employing a biotin modified CMP-sialic acid derivative. We found that this approach has exceptional efficiency compared to two-step SEEL or metabolic labeling, which greatly improved the ability to enrich and identify large numbers of tagged glycoproteins by LC−MS/MS. The one-step approach offers exciting possibilities to study the trafficking and identification of subsets of cell surface glycoconjugates with unprecedented sensitivity in whole cells. Furthermore, we explored a new two-step SEEL, which takes advantage of a sugar nucleotide functionalized by tetrazine, and fast bioorthogonal reaction-DARinv (Inverse electron demand Diels–Alder reaction). This approach will first remodel the cell surface with high reactive tetrazines, followed by click reaction with trans-cyclooctenes (TCO) modified functionalities. This new approach has comparable efficiency with one-step SEEL benefit by the fast click reaction, but has broader applications. We prepared well-defined anti-CD22 ADCs through DARinv and SPANC (Strained promoted alkyne-nitrone cycloaddition) by using tetrazine and nitrone modified CMP-sialic acid derivatives, and the formed ADCs showed dose-dependent cytotoxicity. Furthermore, DARinv and SPANC are orthogonal, which can be used together on the same mAb to deliver two different drugs to increase ADC efficacy and reduce toxicity at the same time.