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.

Enhancement of dendritic cell antigen cross-presentation by CpG DNA involves type I IFN and stabilization of class I MHC mRNA.

Dendritic cells (DCs) internalize exogenous Ags and process them for cross-presentation by class I MHC (MHC-I) to CD8+ T cells. This processing can occur by transporter for Ag presentation (TAP)-dependent or TAP-independent mechanisms. We observed that CpG DNA enhanced cross-presentation of Ags by Flt-3L-cultured bone marrow-derived murine DCs by a type I IFN (IFN-alphabeta)-dependent mechanism. Myeloid DCs provided cross-presentation function in this system. Both TAP1 knockout and wild-type DCs showed enhanced cross-presentation when treated with CpG DNA at 26 degrees C, demonstrating that TAP is not essential to this regulatory mechanism, although TAP is an important determinant of MHC-I expression. Enhancement of cross-processing by CpG DNA did not involve increased Ag uptake or proteolysis but did correlate with IFN-alphabeta-dependent increases in expression of MHC-I mRNA and protein. Increased MHC-I mRNA levels resulted in part from stabilization of MHC-I mRNA, a novel posttranscriptional mechanism for regulation of MHC-I expression. Thus, a major mechanism by which CpG oligodeoxynucleotide increase cross presentation by DCs appears to be an IFN-alphabeta-mediated increase in MHC-I synthesis.

Synthesis and immunological properties of N-modified GM3 antigens as therapeutic cancer vaccines.

The problem of immunotolerance to GM3, an important tumor-associated trisaccharide antigen, seriously hinders its usage in cancer vaccine development. To solve this problem, the keyhole limpet hemocyanin (KLH) conjugates of a series of GM3 derivatives were synthesized and screened as therapeutic cancer vaccines. First, the beta-linked anomeric azides of differently N-acylated GM3 analogues were prepared by a highly convergent procedure. Next, a pentenoyl group was linked to the reducing end of the carbohydrate antigens following selective reduction of the azido group. The linker was thereafter ozonolyzed to give an aldehyde functionality permitting the conjugation of the antigens to KLH via reductive amination. Finally, the immunological properties of the resultant glycoconjugates were studied in C57BL/6 mice by assessing the titers of specific antibodies induced by the GM3 analogues. While KLH-GM3 elicited low levels of immune response, the KLH conjugates of N-propionyl, N-butanoyl, N-iso-butanoyl, and N-phenylacetyl GM3s induced robust immune reactions with antibodies of multiple isotypes, indicating significantly improved and T-cell dependent immune responses that lead to isotype switching, affinity maturation, and the induction of immunological "memory". It was suggested that GM3PhAc-KLH is a promising vaccine candidate for glycoengineered immunotherapy of cancer with GM3 as the primary target.

Preparation and immunological studies of protein conjugates of N -acylneuraminic acids.

The overexpression of N -acetylneuraminic acid (Neu5Ac) is closely correlated with malignant transformations. Thus, Neu5Ac is an important target in the design of cancer vaccines. To study the influence of chemical modifications of Neu5Ac on its immunological properties, the alpha-allyl glycosides of five differently N -acylated neuraminic acid derivatives were prepared. Following selective ozonolysis of their allyl group to form an aldehyde functionality, they were coupled to keyhole limpet hemocyanin (KLH) via reductive amination. Resultant glycoconjugates were studied in C57BL/6 mice. The N -propionyl, N - iso- butanoyl and N -phenylacetyl derivatives of neuraminic acid provoked robust immune responses of various antibody isotypes, including IgM, IgG1, IgG2a and IgG3, whereas N -trifluoropropionylneuraminic acid and natural Neu5Ac were essentially nonimmunogenic. Moreover, the N -phenylacetyl and N - iso- butanoyl derivatives mainly induced IgG responses that are desirable for antitumor applications. These results raise the promise of formulating effective glycoconjugate cancer vaccines via derivatizing sialic acid residues of sialooligosaccharides.

Tapasin-/- and TAP1-/- macrophages are deficient in vacuolar alternate class I MHC (MHC-I) processing due to decreased MHC-I stability at phagolysosomal pH.

Alternate class I MHC (MHC-I) Ag processing via cytosolic or vacuolar pathways leads to cross-presentation of exogenous Ag to CD8 T cells. Vacuolar alternate MHC-I processing involves phagolysosomal Ag proteolysis and peptide binding to MHC-I in post-Golgi compartments. We report the first study of alternate MHC-I Ag processing in tapasin(-/-) cells and experiments with tapasin(-/-) and TAP1(-/-) macrophages that characterize alternate MHC-I processing. Tapasin promotes retention of MHC-I in the endoplasmic reticulum (ER) for loading with high affinity peptides, whereas tapasin(-/-) cells allow poorly loaded MHC-I molecules to exit the ER. Hypothetically, we considered that a large proportion of post-Golgi MHC-I on tapasin(-/-) cells might be peptide-receptive, enhancing alternate MHC-I processing. In contrast, alternate MHC-I processing was diminished in both tapasin(-/-) and TAP1(-/-) macrophages. Nonetheless, these cells efficiently presented exogenous peptide, suggesting a loss of MHC-I stability or function specific to vacuolar processing compartments. Tapasin(-/-) and TAP1(-/-) macrophages had decreased MHC-I stability and increased susceptibility of MHC-I to inactivation by acidic conditions (correlating with vacuolar pH). Incubation of tapasin(-/-) or TAP1(-/-) cells at 26 degrees C decreased susceptibility of MHC-I to acid pH and reversed the deficiency in alternate MHC-I processing. Thus, tapasin and TAP are required for MHC-I to bind ER-derived stabilizing peptides to achieve the stability needed for alternate MHC-I processing via peptide exchange in acidic vacuolar processing compartments. Acidic pH destabilizes MHC-I, but also promotes peptide exchange, thereby enhancing alternate MHC-I Ag processing. These results are consistent with alternate MHC-I Ag processing mechanisms that involve binding of peptides to MHC-I within acidic vacuolar compartments.