| Popis: |
Tumor associated carbohydrate antigens (TACAs) are aberrantly expressed glycan structures on the surface of cancer cells and exist as a downstream result of mutations involved with glycosyltransferases and chaperone proteins. The ubiquitous expression of TACAs on epithelial cancers creates an avenue for immune intervention. However, carbohydrate antigens are T cell independent and consequently lack the ability to induce immunological memory and antibody isotype switching. These two features of the adaptive arm of immunity are pivotal in the development of successful immune responses. A classical strategy to evade this issue requires the use of TACA-glycoconjugates which incorporate both a TACA and an immunologically relevant molecule to trick the immune system to induce T cell dependent immunity towards these glycan antigens. Seminal work in this field has utilized proteins as the immunological carrier, but contemporary research involves the investigation of other non-protein sources such as zwitterionic polysaccharides (ZPS). This classification of immunogenic hapten carriers have emerged after their discovery to induce CD4+ T cell dependent immune responses. The most extensively studied ZPS, PS A1, is a constituent of the bacterial capsule of Bacteroides fragilis (ATCC® 25285™/NTTC® 9343™). PS A1 is composed of a tetrasaccharide repeating unit equipped with alternating charges on adjacent monosaccharides, a feature integral to its immunogenic character. We have utilized this unique ZPS to create therapeutic cancer vaccine candidates as well as to develop a monoclonal antibody. Initial investigations of this strategy by the Andreana group have focused on the mucin related TACAs such as the Thomsen-nouveau (Tn), Thomsen-Friedenreich (TF), and Sialyl Thomsen-nouveau (STn) antigens. These specific TACA-PS A1 conjugates were demonstrated to induce robust immunity in Jax C57BL/6 mice and were evaluated for antibody production, antibody function, and cytokine production. The initial results were promising and have led to further investigations of immune mechanisms such as the quantification of cytokine producing cells. This investigation utilized the ELISpot technique to quantify splenocytes from immunized Jax C57BL/6 mice in vitro that produced cytokines such as IFN-γ, IL-10, and IL-17A. These results definitively shown T cell activation with these PS A1 conjugates that were specific and significantly enhanced as compared to PS A1 alone. Additionally, these investigations lead to the development of a bivalent, unimolecular design to enhance an immune response towards the TF antigen. This design incorporated both the Tn and the TF antigen on PS A1 in which Tn was hypothesized to act as a ligand for the C-type lectin receptor (CLR) Macrophage Galactose N-acetyl Galactosamine Specific Lectin 2 (MGL2). This strategy could enhance immune activation by an increased valency of interaction on antigen presenting cells (APCs) and to alter intracellular signalling to induce increased amounts of pro-inflammatory cytokines that result in a more protective phenotype. The progress of these initial developments led us to expand our vaccine platform to incorporate the ganglioside GM3. GM3 is overexpressed in carcinomas of the breast and skin and has implications in tumor cell proliferation and metastasis. Tumor malignancy is correlated with GM3 expression with high levels of GM3 leading to poor prognosis. Due to these characteristics, GM3 has been prioritized within the top 50 cancer antigens for immune intervention. An aminooxy GM3 trisaccharide derivative was chemically synthesized using a linear glycosylation approach. The key step in this synthesis involved a highly alpha selective sialylation to a galactose acceptor as opposed to less alpha resolving lactose acceptors. The disaccharide intermediate was then functionalized as a donor for the [2 + 1] glycosylation with a glucose acceptor equipped with an O-succinimidyl (-OSu) group on its reducing end. The fully deprotected aminooxy GM3 derivative was then conjugated to the immunologically relevant PS A1 via oxime ligation to generate the vaccine candidate GM3-PS A1.The Andreana group has also demonstrated the utility of PS A1 conjugates with the production of a monoclonal IgM antibody that targets the Tn antigen. Monoclonal antibodies (mAbs) have emerged in the pharmaceutical realm as viable and profitable agents to fight disease, and only a handful of these emerged mAbs target glycan antigens. With only one of these glycan specific mAbs gaining FDA approval (i.e. Unituxin®), much effort has been focused on generating mAbs with higher function and specificity. Furthermore, IgG mAbs are readily investigated due to their relatively more specific nature and their stability during common lab purification techniques. IgM mAbs are emerging as promising therapeutic candidates due to their increased avidity and their enhanced ability to activate compliment. The IgM isotype is typically expressed as a pentamer making IgM antibodies the largest isotype by molecular weight, and consequently IgM antibodies are the most labile isotype during common purification techniques. As purification is necessary to fully characterize its properties, we elected the use of affinity chromatography with mannose binding protein (MBP). The purified IgM mAb was then analyzed for specificity on a glycan array which yielded positive results of preferential binding to the Tn antigen. This binding was demonstrated on a monovalent Tn structure. |
