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Chemical Synthesis, Immunologic Evaluation and Conformational Analysis by NMR Spectroscopy of Complex Glycoconjugate Adjuvants for Anti-Cancer and Anti-Viral Vaccine Therapies

Final Report Summary - VACCINE ADJUVANTS (Chemical Synthesis, Immunologic Evaluation and Conformational Analysis by NMR Spectroscopy of Complex Glycoconjugate Adjuvants for Anti-Cancer and Anti-Viral Vaccine Therapies)


Adjuvants are substances that enhance the immune response and are added to vaccines to make them work better. They increase the immunogenicity of the antigen, enabling the dose-sparing of rare and otherwise impotent antigens, and also extend the immunotherapeutic benefits of vaccination to poor responders, such as children and the elderly. Unfortunately, only a limited number of immunological adjuvants that meet the requirements of potent activity and acceptable toxicity are available for use in vaccines. QS-21, a member of the naturally occurring class of compounds known as saponins, is one of the most promising immunoadjuvants and, structurally, consists of four principal domains: a branched trisaccharide, a central triterpene core, a linear tetrasaccharide and a glycosylated acyl side chain. It has been investigated in numerous vaccine clinical trials against cancer and infectious diseases, including the recently developed Mosquirix malaria vaccine. However, there are several liabilities associated with the natural product that impede its further commercial advancement; namely, limited availability and heterogeneity, adverse side effects, and chemical instability due to the presence of easily hydrolyzable ester linkages in its acyl chain. On top of this, the mechanism of action of QS-21 remains enigmatic, making difficult to create enhanced versions of this compound in a rationally designed way. The late Prof. David Gin and co-workers at Memorial Sloan Kettering Cancer Center (New York) began to address these challenges by developing the required synthetic technology for the first total synthesis of QS-21 as well as a semi-synthetic approach for the preparation of stable analogues incorporating simplified acyl chains with more robust amide linkages. The “Vaccine Adjuvants” project continued to pursue the goal of developing structurally simpler and improved QS-21 variants that can also be utilized for the investigation of their mechanisms of immunopotentiation.

Initially, we synthesized and immunologically evaluated a second-generation of amide-derived acyl chain analogues bearing diverse, alternative moieties at this domain, as well as linear tetrasaccharide variants via systematic truncation of this domain. Preclinical evaluation in mice followed our established mouse vaccination model and involved a mixture of antigens (GD3-KLH, MUC1-KLH, and OVA) of different immunogenicities co-administered with the saponin adjuvant candidate in three weekly subcutaneous injections plus a fourth, booster immunization. The early work identified a linear trisaccharide variant having a terminal carboxylic acid group at the acyl chain that was equipotent to QS-21 but offered improved synthetic accessibility and water solubility. Although this variant exhibited some toxicity, further saccharide truncation resulted in decreased adjuvant activity; the corresponding disaccharide was also more toxic and the monosaccharide derivative was essentially inactive. On the other hand, while the presence of a primary amine at the acyl chain terminus in the trisaccharide series abrogated activity, its acylation could, in some cases, restore adjuvant activity, providing a versatile scaffold for derivatization with different probes (e.g. fluorophores or radiolabels) for subsequent mechanistic studies.

Next, we investigated the influence of the left-hand branched trisaccharide domain in adjuvant activity and toxicity with the synthesis of a truncated variant devoid of this domain that also incorporated an aryl iodide at the acyl chain terminus with a view towards developing radioiodinated probes for biodistribution studies. Strikingly, this new analogue elicited antibody responses comparable to QS-21, retaining QS-21’s adjuvant activity while also lowering its toxicity. The dispensability of the branched trisaccharide for activity enabled us to explore additional truncated variants with targeted modifications in the triterpene domain by using other readily available triterpenes as starting materials. Chemical synthesis and immunological evaluation of a new, echinocystic acid variant, where the triterpene C4-aldehyde substituent is replaced with a methyl group, revealed that, in contrast to previous hypothesis in the literature, this aldehyde is not required for adjuvant activity in our synthetic saponins. In addition, this novel saponin adjuvant exhibited antibody responses rivalling those of QS-21 without its associated toxicity, which makes it a promising lead for further development towards clinical evaluation. We also showed that the small molecule immunopotentiator tucaresol, which also has an aldehyde, did not enhance adjuvant activity of our saponins, either in combination or incorporated covalently into the acyl chain of these active variants.


Our extensive chemical studies on every domain of QS-21 provided critical information about the structural features required for adjuvant activity, and enabled the development of radiolabeled and fluorescent saponin probes for the first in vivo biodistribution studies of these novel saponins. Use of these radioiodinated and fluorescently-labeled versions revealed that active adjuvants accumulate in the lymph nodes, increase lymph node localization of an antigen and get internalized by dendritic cells, while inactive ones don’t. This positive correlation between adjuvant localization/biodistribution and its immunopotentiating activity suggest that these saponin variants may act by facilitating antigen trafficking by antigen-presenting cells to the lymph nodes, a known site for immune cell maturation, where the processed antigens are presented to the adaptive immune system.

We have also developed a new, divergent synthetic approach to saponin variants having modifications in specific sugars within the linear oligosaccharide domain. These novel analogues were designed specifically for greater synthetic accessibility and were all prepared in fewer steps than our previous lead compound. This modular, versatile strategy involved stepwise glycosylation with a single bridging monosaccharide followed by subsequent attachment of a variety of terminal disaccharides, overcoming significant limitations of our original convergent approach, in which the entire trisaccharide had to be assembled separately and then coupled en bloc to the triterpene domain. The efficiency and versatility of this divergent route will enable rapid access to a wide range of linear oligosaccharide variants to identify novel saponin immunoadjuvants that are more synthetically accessible and scalable for clinical advancement in vaccines.

Most recently, we have performed the conformational analysis of QS-21 variants with modifications at the central glycosidic linkage that connects the triterpene and linear oligosaccharide domains. Relatively conservative structural changes at this junction resulted in striking modulation of in vivo adjuvant activities, which led us to study the implications of such modifications on saponin conformation. Exploration of the structural behaviour and dynamic features of these saponin variants revealed that adjuvant-active saponins, including QS-21, exhibit relatively rigid structures where the acyl chain domain is folded back over the triterpene and the linear trisaccharide adopts an extended orientation away from the center of the triterpene. However, the acyl chain and trisaccharide domains in inactive variants were more disorganized and exhibited different relative orientations, suggesting the existence of a preferred, folded “active conformation” for the potent adjuvants. Thus, our conformational studies identified a correlation between three-dimensional structure and adjuvant activity, providing a molecular rational for the divergent adjuvant activities observed in vivo.

In summary, the “Vaccine Adjuvants” project has provided access to saponin adjuvants that are potent, non-toxic and easier to make, while yielding new, critical cellular and molecular insights into saponin mechanisms of action. These results fill a gap in knowledge in the vaccine adjuvant field, open new avenues for adjuvant research, and promise to enable rational development of optimized adjuvant-antigen combinations for a wide range of vaccines, further highlighting the impact and significance of the research carried out in this successful project.