Efficient metabolic engineering of GM3 on tumor cells by N-phenylacetyl-D-mannosamine

Abnormal carbohydrates expressed on tumor cells, which are termed tumor-associated carbohydrate antigens (TACAs), are potential targets for the development of cancer vaccines. However, immune tolerance to TACAs has severely hindered progress in this area. To overcome this problem, we have developed a novel immunotherapeutic strategy based on synthetic cancer vaccines and metabolic engineering of TACAs on tumor cells. One critical step of this new strategy is metabolic engineering of cancer, namely, to induce expression of an artificial form of a TACA by supplying tumors with an artificial monosaccharide precursor. To identify the proper precursor for this application, N-propionyl, N-butanoyl, N-isobutanoyl, and N-phenylacetyl derivatives of d-mannosamine were synthesized, and their efficiency as biosynthetic precursors in modifying sialic acid and inducing expression of modified forms of GM3 antigen on tumor cells was investigated. For this purpose, tumor cells were incubated with different N-acyl-d-mannosamines, and modified forms of GM3 expressed on tumor cells were analyzed by flow cytometry using antigen-specific antisera. N-Phenylacetyl-d-mannosamine was efficiently incorporated in a time- and dose-dependent manner to bioengineer GM3 expression by several tumor cell lines, including K562, SKMEL-28, and B16-F0. Moreover, these tumor cell lines also exhibited ManPAc-dependent sensitivity to cytotoxicity mediated by anti-PAcGM3 immune serum and complement. These results provide an important validation for this novel therapeutic strategy. Because N-phenylacetyl GM3-protein conjugates are particularly immunogenic, the combination of an N-phenylacetyl GM3 conjugate vaccine with systemic N-phenylacetyl-d-mannosamine treatment is a promising immunotherapy for future development and application to melanoma and other GM3-bearing tumors.