N-Carboxyanhydride Polymerization of Immunomodulatory Glycopolypeptides for Cancer Immunotherapy

Glycopolymers have served as useful tools for studying glycobiology for over two decades. A significant body of literature has demonstrated that these macromolecules are capable of predictably stimulating lectin receptors, suggesting a potential for clinical applications ranging from adjuvants to immunosuppressants. However, commonly-used scaffolds exhibit notable limitations, including unnatural backbones and glycosidic linkages, which limit their therapeutic potential. In a recent publication, we demonstrated that N-carboxyanhydride (NCA) polymerization is amenable to glycosylated monomers, allowing for the synthesis of true glycopolypeptides. In this dissertation, I demonstrate the applicability of the platform to a variety of glycan structures. In particular, I focus on potentially immunogenic carbohydrate motifs with the ultimate goal of developing synthetic C-type lectin receptor (CLR) agonists designed to activate antigen-presenting cells (APCs) for cancer immunotherapy./nDesigning such immunostimulatory glycopeptides requires a general understanding of the cells and pathways important to generating an anticancer immune response. In Chapter 1, I provide an overview of APCs and their associated immune receptors, including CLRs. I include in this section a brief summary of reported pattern recognition receptor agonists, including clinically-approved synthetic ligands, and note the lack of well-defined CLR agonists. I then discuss the relationship between cancer and the immune system with the goal of rationalizing our strategy of activating APCs to induce a robust anticancer response. To provide further context, I conclude with a summary of modern and historical approaches to cancer immunotherapy./nOur initial NCA polymerization publication focused on an N-acetylgalactosamine-serine monomer; synthesis of the desired immune-activating glycopolypeptides required that I first establish the generalizability of the platform to other carbohydrates. In Chapter 2, I present work showing that di- and trisaccharide-serine NCAs can be polymerized using transition metal initiators to give glycopolypeptides of controllable length and glycosylation density. In particular, I focus on mannosylated and glucosylated structures that could potentially bind and activate CLRs on antigen-presenting cells, setting the stage for my intended therapeutic application./nIn Chapter 3, I demonstrate for the first time that NCA-derived glycopolypeptides can be rationally designed to activate desired CLRs. Specifically, glucosylated and mannobiosylated structures stimulate murine monocyte-derived cells and dendritic cells in vitro through the CLRs Dectin-1 and Dectin-2, respectively. Activation was dependent on the carbohydrate structures used and consistent with previous literature describing glycan preferences of these receptors. Chapter 4 expands on these promising results by taking the mannobiosylated glycopolypeptides, which activate the dendritic cell receptor Dectin-2, into murine models of pancreatic cancer. I first show that these materials are able to inhibit tumor growth in vivo. Furthermore, conjugation of these Dectin-2 agonists to a costimulatory Toll-like receptor agonist greatly enhances cytokine release in cell culture and tumor growth inhibition in vivo. Alternatively, attachment of these glycopolypeptides to tumor-targeting antibodies similarly enhances their immunostimulatory effects. These observations provide promising evidence that activation of APCs via NCA-derived glycopolypeptides may indeed be a viable strategy for cancer immunotherapy./nFinally, in Chapter 5 I describe some ongoing challenges with the NCA polymerization technology. As a platform, it would be desirable to have access to glycopolypeptides with multiple functional handles to maximize the available conjugation possibilities. Here, I describe my efforts to improve on our previously-reported dual-end-labeling technology. Through additional characterization and optimization of functionalized NCA initiators, I uncover some flaws with our initial strategy and propose potential solutions. This work points to the need for new initiators for the NCA polymerization of glycopolypeptides. Advancements in this area will allow us to better access multicomponent glycopolypeptide-based conjugates in the future.

Advisor: Bertozzi, Carolyn R
Wemmer, David E