Sialyl Thomsen-nouveau (STn) is a tumor-associated carbohydrate antigen (TACA) that is overexpressed in a variety of carcinomas such as breast, ovarian, and colon cancer. In normal tissue, STn is not detectable, which is critical for opportunities in developing cancer immunotherapies. A novel, entirely carbohydrate, semisynthetic STn-polysaccharide (PS) A1 conjugate was prepared and evaluated in C57BL/6 mice. STn-PS A1 was combined with commercially available monophosphoryl lipid A-based adjuvant, and after immunization, ELISA indicated a strong immune response for inducing anti-STn IgM/IgG antibodies. The specificity of these antibodies was concomitantly investigated using FACS analysis, and the results indicated excellent cell surface binding events to STn-expressing cancer cell lines MCF-7 and OVCAR-5. An INF-γ ELISpot assay was conducted to further confirm a robust cellular immunity invoked by STn-PS A1. Most importantly, the raised antibodies conferred complement-dependent cellular cytotoxicity against MCF-7 and OVCAR-5 cells.

We have developed a novel antigen delivery system based on polysaccharide-coated gold nanoparticles (AuNPs) targeted to antigen-presenting cells (APCs) expressing Dectin-1. AuNPs were synthesized de novo using yeast-derived β-1,3-glucans (B13G) as the reductant and passivating agent in a microwave-catalyzed procedure, yielding highly uniform and serum-stable particles. These were further functionalized with both a peptide and a specific glycosylated form from the tandem repeat sequence of mucin 4 (MUC4), a glycoprotein overexpressed in pancreatic tumors. The glycosylated sequence contained the Thomsen–Friedenreich disaccharide, a pan-carcinoma, tumor-associated carbohydrate antigen (TACA), which has been a traditional target for antitumor vaccine design. These motifs were prepared with a cathepsin B protease cleavage site (Gly-Phe-Leu-Gly), loaded on the B13G-coated particles, and these constructs were examined for Dectin-1 binding, APC processing, and presentation in a model in vitro system and for immune responses in mice. We showed that these particles elicit strong in vivo immune responses through the production of both high-titer antibodies and priming of antigen-recognizing T-cells. Further examination showed that a favorable antitumor balance of expressed cytokines was generated, with limited expression of immunosuppressive Il-10. This system is modular in that any range of antigens can be conjugated to our particles and efficiently delivered to APCs expressing Dectin-1.

The Thomsen-Friedenreich (TF) antigen is a key target for the development of anticancer vaccines, and this ongoing challenge remains relevant due to the poor immunogenicity of the TF antigen. To overcome this challenge, we adopted a bivalent conjugate design which introduced both the TF antigen and the Thomsen-nouveau (Tn) antigen onto the immunologically relevant polysaccharide A1 (PS A1). The immunological results in C57BL/6 mice revealed that the bivalent, Tn-TF-PS A1 conjugate increased the immune response towards the TF antigen as compared to the monovalent TF-PS A1. This phenomenon was first observed with enzyme-linked immunosorbent assay (ELISA) where the bivalent conjugate generated high titers of IgG antibodies where the monovalent conjugate generated an exclusive IgM response. Fluorescence-activated cell sorting (FACS) analysis also revealed increased binding events to the tumor cell lines MCF-7 and OVCAR-5, which are consistent with the enhanced tumor cell lysis observed in a complement dependent cytotoxicity (CDC) assay. The cytokine profile generated by the bivalent construct revealed increased pro-inflammatory cytokines IL-17 and IFN-γ. This increase in cytokine concentration was matched with an increase in cytokine producing cells as observed by ELISpot. We hypothesized the mechanisms for this phenomenon to involve the macrophage galactose N-acetylgalactosamine specific lectin 2 (MGL2). This hypothesis was supported by using biotinylated probes and recombinant MGL2 to measure carbohydrate-protein interactions.