Structure-function characterization and engineering of polysaccharides and antibodies with therapeutic activity

摘要

Proteins and polysaccharides are of growing importance as a source for novel therapeutic compounds and target a range of diseases, from cancer to infections from pathogens. However, owing to their large and complex structures, they face a unique set of challenges, compared to small molecules, in their discovery and development as safe, efficacious drugs. Towards addressing these challenges, we describe in this thesis the implementation of structure-function relationship approaches to characterize and engineer polysaccharides and antibodies to improve their therapeutic profiles. The plant polysaccharide pectin, when modified, has demonstrated significant anticancer activity in animal models and small-scale clinical trials. Its development has been hampered, however, due to its complex structure and lack of structure-activity correlates. Using an integrated approach, we engineer a modified pectin that exhibits significant in vivo anticancer activity, which we link to specific structural attributes and cellular functional mechanisms. These results improve our structure-function understanding of anticancer modified pectin, an important step towards the clinical use of this complex polysaccharide. Applying what we learned from pectin, we develop an integrated framework to identify a contaminant in batches of heparin, a polysaccharide anticoagulant drug, associated with an outbreak of allergic-type reactions in 2007-2008. Employing orthogonal analytical approaches to overcome challenges of characterizing structurally complex pharmaceutical heparin, we determine that the structurally related glycan, oversulfated chondroitin sulfate, is the major contaminant. We link its presence to activation of the contact pathway, thereby establishing a structure-function understanding of contaminated heparin and improving the safety profile of this polysaccharide drug. Transitioning knowledge gained from the structure-function characterization of polysaccharides, we engineer, by structure-based design, a broad spectrum neutralizing antibody to dengue virus, which yearly infects more than 200 million people, causing approximately 21,000 deaths. We incorporate complementary approaches of energetics and empirical informatics methods to rationally redesign an existing antibody for greater breadth and potency, resulting in an engineered antibody with binding to all four virus serotypes and good in vitro potency. Overall, this thesis provides important insights into structure-function approaches through the use of complementary methods to characterize and engineer therapeutic polysaccharides and antibodies.