Abstract
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.
Highlights
The Thomsen-Friedenreich (TF, β-d-Galp-(1,3)-α-d-GalNAcp) antigen is a tumor associated carbohydrate antigen (TACA) that has significant roles in the progression of carcinomas of the breast, colon, prostate, liver, and more [1]
To avoid oxidative cleavage of the furanose ring, 0.5 equivalents of sodium periodate was used per repeating unit of polysaccharide A1 (PS A1) to ensure regioselectivity
Tn-PS A1 was observed to be consistent in mounting an IgG immune response towards the Tn antigen whether TiterMax Gold (TMG) or Sigma Adjuvant System (SAS) was used
Summary
The Thomsen-Friedenreich (TF, β-d-Galp-(1,3)-α-d-GalNAcp) antigen is a tumor associated carbohydrate antigen (TACA) that has significant roles in the progression of carcinomas of the breast, colon, prostate, liver, and more [1]. High expression levels of the TF antigen on tumor cells is positively correlated to poor prognosis, and an increased ability to metastasize [2,3]. The relationship between TF expression and metastasis has been definitively shown to be mediated through galectin-3 on endothelial cells, where selective pressure to non-TF expressing tumor populations can decrease metastasis [4,5]. Patients that were able to develop endogenous anti-TF antibodies have significantly improved prognosis [6,7] These characteristics of the TF antigen coupled to the fact that the TF antigen is not exposed on normal, healthy tissue makes the TF antigen a viable target for immunotherapy [8,9].
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