Effective Sugar Nucleotide Regeneration for the Large-Scale Enzymatic Synthesis of Globo H and SSEA4 Tsung-I Tsai, †,‡ Hsin-Yu Lee, †,§ Shih-Huang Chang, †,∥ Chia-Hung Wang, † Yu-Chen Tu, † Yu-Chen Lin, † Der-Ren Hwang, † Chung-Yi Wu, † and Chi-Huey Wong* ,†,‡ † Genomics Research Center, Academia Sinica, No. 128, Section 2, Academia Road, Taipei 115, Taiwan ‡ Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan § Department of Chemistry and ∥ Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan * S Supporting Information ABSTRACT: We report here the development of chemo- enzymatic methods for the large-scale synthesis of cancer- associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyl- transferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics. ■ INTRODUCTION Glycosphingolipids play important roles in many physiological conditions, including development and differentiation, tumor progression, cell adhesion, and signal transduction. Among the glycosphingolipids, the globo series of gangliosides is strongly associated with malignant diseases such as breast, lung, ovary, stomach, and small-cell lung cancers but is not detectable in normal cells. 1−4 For example, Globo H was first discovered in 1983 from a cultured human teratocarcinoma cell line 5,6 and then from a variety of epithelial tumors such as colon, ovarian, gastric, pancreatic, endometrial, lung, prostate, and breast cancers by staining with the monoclonal antibodies MBr1 and VK9. 7−10 However, the biological function of this molecule is not clear, though its expression is associated with tumor aggressiveness in breast carcinoma, small-cell lung carcinoma, and several malignant cancers. 11,12 These studies have led to the development of a therapeutic cancer vaccine based on synthetic Globo H conjugated to keyhole limpet hemocyanin (KLH) and adjuvant QS-21 for the treatment of breast cancer and prostate cancer, and positive results were observed in phase I clinical trials. 13−15 This vaccine was further advanced to phase III clinical trials for breast cancer with improved synthesis of Globo H by the programmable one-pot method. 16 Our previous studies by flow cytometry also revealed that Globo H was overexpressed in ∼61% of breast cancer patients and Gb5 was overexpressed in ∼77.5% of patients 17 and glycosphingolipids Gb5, Globo H, and SSEA4 were found on breast cancer cells and the cancer stem cells but were not detectable on normal cells. 18 In addition, glycan array analysis demonstrated that higher levels of anti-Globo H antibody exist in the plasma of breast cancer patients. 19,20 Compared to the breast cancer target HER2 that was present in 20% of breast cancer patients, 21 the glycosphingolipid antigens (i.e., Gb5 and its derivatives, Globo H and SSEA4) were much more predominant and are thus better candidates for the develop- ment of cancer vaccines or therapeutic antibodies. To improve the vaccine efficacy with regard to its immune response, Globo H has been conjugated to different carrier proteins through different linkers and used in combination with different adjuvants in animal studies. 18 It was found that when Globo H was conjugated to CRM197 (diphtheria toxin) and used in combination with the glycolipid adjuvant C34, the vaccine elicited a significant IgG response to recognize not only Globo H but also Gb5 and SSEA4, as compared to the Globo H-KLH- QS21 vaccine that elicited a significant IgM antibody response with less selectivity toward tumor-associated glycans. 18 Currently, the synthesis of complex carbohydrates for clinical study is available by chemical methods; however, it has been a challenge in clinical development because of the lack of methods available for large-scale synthesis. 22 The traditional chemical synthesis is tedious and labor-intensive because the complex protection, deprotection, and extensive purification steps are necessary to achieve high purity and structural integrity. The problem has stimulated the development of an enzymatic method. 23 Nevertheless, several methods have been Received: July 23, 2013 Published: September 17, 2013 Article pubs.acs.org/JACS © 2013 American Chemical Society 14831 dx.doi.org/10.1021/ja4075584 | J. Am. Chem. Soc. 2013, 135, 14831−14839