␣-Galactosylceramide (␣-GalCer), a glycolipid that stimulates natural killer T (NKT) cells to produce both T helper (Th)1 and Th2 cytokines, has shown antitumor effects in mice but failed in clinical trials. We evaluated 16 analogs of ␣-GalCer for their CD1-mediated T cell receptor (TCR) activation of naïve human NKT cells and their anticancer efficacy. In vitro, glycolipids containing an aromatic ring in their acyl tail or sphingosine tail were more effective than ␣-GalCer in inducing Th1 cytokines/chemokines, TCR activation, and human NKT cell expansion. None of these glycolipids could directly stimulate human dendritic cell maturation, except for a glycolipid with an aromatic ring on the sphingosine tail. Here, we show that glycolipids activated the TCR of NKT cells with phosphorylation of CD3, ERK1/2, or CREB, which correlated with their induction of Th1 cytokines. Notably, the extent of NKT cell activation when glycolipid was presented by antigen-presenting cells was greater than when glycolipid was presented by non-antigenpresenting cells. In vivo, in mice bearing breast or lung cancers, the glycolipids that induced more Th1-biased cytokines and CD8/CD4 T cells displayed significantly greater anticancer potency than ␣-GalCer. These findings indicate that ␣-GalCer analogs can be designed to favor Th1-biased immunity, with greater anticancer efficacy and other immune-enhancing activities than ␣-GalCer itself.antigen presentation ͉ CD1 ͉ ␣-galactosylceramide

We examined the expression in breast cancer stem cells (BCSCs) of Globo H, a potential tumor-associated antigen for immunotherapy of epithelial cancers including breast cancer. Flow cytometry revealed Globo H expression in 25/41 breast cancer specimens (61.0%). Non-BCSCs from 25/25 and BCSCs from 8/40 (20%) expressed Globo H. We showed the expression of stage-specific embryonic antigen 3 (SSEA3), the pentasaccharide precursor of Globo H, in 31/40 (77.5%) tumors. Non-BCSCs from 29/31 and BCSCs from 25/40 (62.5%) expressed SSEA3. Like Globo H, SSEA3 expression in normal tissues was predominately at the secretory borders of epithelium, where access to the immune system is restricted. Immunization of mice with Globo H-KLH and α-GalCer induced antibodies reactive with Globo H and SSEA3, suggesting that a Globo H-based vaccine will target tumor cells expressing Globo H or SSEA3. We next sought to reduce Globo H expression by siRNA targeting fucosyltransferase ( FUT ) 1 and 2, which mediate alpha-1,2 linkage of fucose to SSEA3 to generate Globo H. We showed both genes to be involved in the biosynthesis of Globo H. Moreover, FUT2 expression in BCSCs was significantly lower than in non-BCSCs harvested from a primary human breast cancer in NOD/SCID mouse, whereas FUT1 was slightly lower in BCSCs. Thus, the lower expression of Globo H in BCSCs may be attributed to less FUT2/FUT1, and to reduced SSEA3 in BCSCs compared with non-BCSCs. Our findings provide insight into further development of a Globo H-based vaccine and FUT1/FUT2 -targeted therapy for breast cancer.

Globo H (GH) is a hexasaccharide specifically overexpressed on a variety of cancer cells and therefore, a good candidate for cancer vaccine development. To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked GH to a carrier protein, including keyhole limpet hemocyanion, diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid, and BSA, and combined with an adjuvant, and it was administered to mice for the study of immune response. Glycan microarray analysis of the antiserum obtained indicated that the combination of GH-DT adjuvanted with the α-galactosylceramide C34 has the highest enhancement of anti-GH IgG. Compared with the phase III clinical trial vaccine, GHkeyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GHrelated epitopes, stage-specific embryonic antigen 3 (SSEA3) and SSEA4, all of which were specifically overexpressed on breast cancer cells and breast cancer stem cells with SSEA4 at the highest level (>90%). We, therefore, further developed SSEA4-DT/C34 as a vaccine candidate, and after immunization, it was found that the elicited antibodies are also IgG-dominant and very specific for SSEA4.carbohydrate vaccine | diphtheria toxin

Cancer-associated carbohydrate antigens are often found on the surface of cancer cells. Understanding their roles in cancer progression will lead to the development of new therapeutics and high-sensitivity diagnostics for cancers. Globo H is a member of this family, which is highly expressed on breast cancer cells. Here, we report the development of a glycan microarray of Globo H and its analogs for measurement of the dissociation constants on surface (K D,surf) with three different monoclonal antibodies (VK-9, Mbr1, and anti-SSEA-3), to deduce their binding specificity. The glycan microarray was also used to detect the amount of antibodies present in the plasma of breast cancer patients and normal blood donors. It was shown that the amount of antibodies against Globo H from breast cancer patients were significantly higher than normal blood donors, providing a new tool for possible breast cancer diagnosis. Compared with the traditional ELISA method, this array method required only atto-mole amounts of materials and is more effective and more sensitive (5 orders of magnitude). The glycan microarray thus provides a new platform for use to monitor the immune response to carbohydrate epitopes after vaccine therapy or during the course of cancer progression.array ͉ diagnosis

Tumor angiogenesis is a critical element of cancer progression, and strategies for its selective blockade are still sought. Here, we examine the angiogenic effects of Globo-H ceramide (GHCer), the most prevalent glycolipid in a majority of epithelial cancers and one that acts as an immune checkpoint. Here, we report that GHCer becomes incorporated into endothelial cells through the absorption of microvesicles shed from tumor cells. In endothelial cells, GHCer addition induces migration, tube formation, and intracellular Ca 2þ mobilization in vitro and angiogenesis in vivo. Breast cancer cells expressing high levels of GHCer displayed relatively greater tumorigenicity and angiogenesis compared with cells expressing low levels of Globo-H. Clincally, GHCer þ breast cancer specimens contained higher vessel density than GHCer À breast cancer specimens. Mechanistic investigations linked the angiogenic effects of GHCer to its endocytosis and binding to TRAX, with consequent release of PLCb1 from TRAX to trigger Ca 2þ mobilization. Together, our findings highlight the importance of GHC as a target for cancer therapy by providing new information on its key role in tumor angiogenesis. Cancer Res; 74(23); 6856-66. Ó2014 AACR.